Stem-loop compositions and methods for inhibiting factor d

ABSTRACT

The application discloses methods and compositions for the inhibition of the alternative complement pathway. The methods and compositions involve the use of aptamers for inhibiting complement Factor D. The application further provides anti-Factor D aptamers for the treatment of dry age-related macular degeneration, geographic atrophy, wet age-related macular degeneration or Stargardt disease. In some cases, stem-loop aptamers are provided for the inhibition of Factor D.

CROSS-REFERENCE

This application is a continuation application of International PatentApplication No. PCT/US2018/014573, filed Jan. 19, 2018, which claims thebenefit of U.S. Provisional Application Nos. 62/448,872, filed Jan. 20,2017, and 62/536,387, filed Jul. 24, 2017, which applications areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 6, 2018, isnamed 49644-716_601_SL.txt and is 100,140 bytes in size.

BACKGROUND OF THE INVENTION

Visual impairment is a national and global health concern that has anegative impact on physical and mental health. The number of people withvisual impairment and blindness is increasing due to an overall agingpopulation. Visual impairment and blindness can be caused by any one ofa large number of eye diseases and disorders affecting people of allages. In one example, age-related macular degeneration (AMD) is an eyedisorder that is currently the leading cause of vision loss in peoplefifty years of age or older in industrialized countries. It is estimatedthat by 2020, the number of people with AMD could exceed 196 million andby 2040, that number is expected to rise to 288 million. AMD is adegenerative eye disease that progresses from early stages to advancedstages of the disease. Risk factors for the disease include aging,lifestyle factors such as smoking, and genetics. The clearest indicatorof progression to AMD is the appearance of drusen, yellow-white depositsunder the retina, and it is an important component of both forms of AMD:exudative (“wet”) and non-exudative (“dry”). Wet AMD causes vision lossdue to abnormal blood vessel growth in the choriocapillaris throughBruch's membrane. The most advanced form of dry AMD, known as geographicatrophy, is generally more gradual and occurs when light-sensitive cellsin the macula atrophy, blurring and eliminating vision in the affectedeye. While there are currently some promising treatments for wet AMD, noFDA-approved treatment exists for dry AMD or geographic atrophy.

A second example is childhood-onset Stargardt Disease (“STGD”), alsoknown as Stargardt 1, a genetic, rare juvenile macular dystrophygenerally associated with loss of central vision in the first twodecades of life. STGD has a prevalence of approximately 1/20,000affecting approximately 30,000 people in the US. STGD affects many ages,with the childhood-onset population at highest risk and most need.Patients with childhood-onset STGD tend to develop early severe visualacuity loss, significantly compromised retinal function, and rapidretinal pigment epithelial (RPE) cell atrophy with accompanying loss ofretinal function. The median ages of onset and the median age atbaseline examination are 8.5 (range, 3-16) and 12 years (range, 7-16),respectively. Patients with adult-onset disease are more likely topreserve visual acuity for a longer time and show slighter retinaldysfunction. STGD is an autosomal recessive genetic disease or complexheterozygous disease, caused by mutations in the ABCA4 gene. The ABCA4gene encodes the photoreceptor protein ABCA4 Transporter, which isresponsible for removal of bisretinoid fluorophores, which can includeN-retinylidene-N-retinyethanolamine (A2E), all-trans-retinal and relatedphoto-oxidation products of vitamin A aldehyde which together constitutelipofuscin from photoreceptor cells. Accumulation of all-trans-retinalin photoreceptor cells is believed to damage RPE cells via oxidativestress, and trigger or promote complement-mediated damage to RPE cells,leading to retinal atrophy. A related disease termed Stargardt-likemacular dystrophy, also known as STGD3, is inherited in a dominantautosomal manner and is due to mutations in the ELOVL4 gene. ELOVL4encodes the ELOVL4 protein, ELOVL fatty acid elongase 4. Mutations inELOVL4 protein associated with STGD lead to mis-folding and accumulationof ELOVL4 protein aggregates in retinal cells, which impact retinal cellfunction, eventually leading to cell death and retinal atrophy. Notreatments exist for STGD or Stargardt-like disease.

SUMMARY OF THE INVENTION

In one aspect, an aptamer is provided comprising a nucleic acid sequencethat selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the third loop comprises 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein the first loop has fewer nucleotides than the second loop.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the third loop comprises 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein the second loop comprises more than 5 nucleotides,non-nucleotidyl spacers, or a combination thereof.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the third loop comprises 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein the third loop is adjacent to the first stem.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the third loop comprises 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein the first base-paired stem has no more than 5 base pairs.

In some cases, the third loop is connected to the first base-pairedstem. In some cases, the first loop has from 1 to 10 nucleotides,non-nucleotidyl spacers, or a combination thereof. In some cases, thefirst loop has from 3 to 5 nucleotides, non-nucleotidyl spacers, or acombination thereof. In some cases, first loop comprises a nucleic acidsequence of 5′-DUG-3′, where D is A, G, or U. In some cases, the secondloop comprises at least 6 nucleotides, non-nucleotidyl spacers, or acombination thereof. In some cases, the second loop comprises at least 7nucleotides, non-nucleotidyl spacers, or a combination thereof. In somecases, the second loop comprises 10 or 11 nucleotides, non-nucleotidylspacers, or a combination thereof. In some cases, the second loopcomprises a nucleic acid sequence of 5′-DWWVGCBHWG-3′ (SEQ ID NO:319),where D is A, G, or U; W is A or U; V is A, C, or G; B is C, G, or U;and H is A, C, or U. In some cases, the second loop comprises a nucleicacid sequence having a U at nucleotide position 2, nucleotide position3, or both. In some cases, the third loop has from 6 to 8 nucleotides,non-nucleotidyl spacers, or a combination thereof. In some cases, thethird loop comprises a nucleic acid sequence comprising 5′-AAGUKN-3′,where K is G or U; and N is A, G, C, or U. In some cases, the firstbase-paired stem has from 2 to 10 base pairs. In some cases, the firstbase-paired stem has from 3 to 8 base pairs. In some cases, the secondbase-paired stem has from 2 to 10 base pairs. In some cases, the secondbase-paired stem comprises 4 or 5 base pairs. In some cases, the secondbase-paired stem comprises a terminal U-G base pair adjacent to thesecond loop. In some cases, the second base-paired stem comprises aterminal C-G base pair adjacent to the second loop. In some cases, thenucleic acid sequence comprises nucleotides having ribose in aβ-D-ribofuranose configuration. In some cases, at least 50% of thenucleic acid sequence comprises nucleotides having ribose in aβ-D-ribofuranose configuration. In some cases, the third loop comprisesat least 4 nucleotides and up to 2 non-nucleotidyl spacers. In somecases, the third loop comprises at least 6 nucleotides. In some cases,the non-nucleotidyl spacers comprise 3 carbons, 6 carbons, or 9 carbons.In some cases, the non-nucleotidyl spacers comprise an 18-atom spacer.In one example, the 18-atom spacer comprises hexaethylene glycol. Insome cases, a) the first base-paired stem is adjacent to said firstloop; b) the second base-paired stem is adjacent to the first loop, thesecond loop, and the third loop; or c) the first base-paired stem isadjacent to the first loop and the second base-paired stem is adjacentto the first loop, the second loop, and the third loop.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising a base-paired terminal stem; anasymmetric internal loop; an internal base-paired stem; and exactly oneterminal loop, wherein the terminal loop comprises more than 4nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein the asymmetric internal loop is adjacent to exactly 2base-paired stems.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the second loop comprises 7 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof,wherein the first base-paired stem has no more than 5 base pairs, andwherein the second base-paired stem comprises more than 2 base pairs.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising exactly one terminalbase-paired stem; exactly one asymmetric internal loop comprising, froma 5′ to 3′ direction, a first loop and a second loop; exactly oneinternal base-paired stem; and exactly one terminal loop, wherein thefirst loop of the asymmetric internal loop has fewer nucleotides thanthe terminal loop.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising exactly one terminalbase-paired stem; exactly one asymmetric internal loop; exactly oneinternal base-paired stem; and exactly one terminal loop, wherein theexactly one terminal loop comprises more than 4 nucleotides,non-nucleotidyl spacers, or a combination thereof. In some cases, theexactly one terminal loop comprises 10 or more nucleotides,non-nucleotidyl spacers, or a combination thereof.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising exactly one terminalbase-paired stem; exactly one asymmetric internal loop comprising, froma 5′ to 3′ direction, a first loop and a second loop; exactly oneinternal base-paired stem; and exactly one terminal loop, wherein thesecond loop comprises 6 or more nucleotides, non-nucleotidyl spacers, ora combination thereof.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising exactly one terminalbase-paired stem; exactly one asymmetric internal loop; exactly oneinternal base-paired stem; and exactly one terminal loop, wherein theexactly one terminal loop comprises 7 or more nucleotides,non-nucleotidyl spacers, or a combination thereof.

In some cases, the exactly one terminal base-paired stem comprises atail at a 5′ end, at a 3′ end, or at both a 5′ end and a 3′ end, and thetail comprises at least one unpaired nucleotide.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the second base-paired stemcomprises a terminal U-G base pair adjacent to the second loop.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the first loop comprises anucleic acid sequence of 5′-DUG-3′, where D is A, G, or U.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the third loop comprises anucleic acid sequence comprising 5′-AAGUKN-3′, where K is G or U; and Nis A, G, C, or U.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the second loop comprises anucleic acid sequence of 5′-DWWVGCBHWG-3′ (SEQ ID NO:319), where D is A,G, or U; W is A or U; V is A, C, or G; B is C, G, or U; and H is A, C,or U.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the second loop comprises anucleic acid sequence having a U at nucleotide position 2, nucleotideposition 3, or both.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein the second base-paired stemcomprises a terminal C-G base pair adjacent to the second loop.

In some cases, any aptamer of the preceding is an RNA aptamer or amodified RNA aptamer. In other cases, any aptamer of the preceding is aDNA aptamer or a modified DNA aptamer. In some cases, any aptamer of thepreceding comprises one or more modified nucleotides. In some instances,at least 50% of the nucleic acid sequence comprises the one or moremodified nucleotides. In some instances, the one or more modifiednucleotides comprises a 2′F-modified nucleotide, a 2′OMe-modifiednucleotide, or a combination thereof. In some instances, the one or moremodified nucleotides are selected from the group consisting of: 2′F-G,2′OMe-G, 2′OMe-U, 2′OMe-A, 2′OMe-C, a 3′ terminal inverteddeoxythymidine, and any combination thereof. In some cases, an aptamerof any of the preceding comprises a nuclease-stabilized nucleic acidbackbone. In some cases, the stem-loop structure of any aptamer of thepreceding has exactly two base-paired stems. In some cases, any aptamerof the preceding is an RNA aptamer comprising nucleotides having ribosein a β-D-ribofuranose configuration. In some cases, any aptamer of thepreceding selectively binds to an active site of fD. In some cases, anyaptamer of the preceding selectively binds to a substrate-bindingexosite of fD. In some cases, any aptamer of the preceding selectivelybinds to both an active site of fD and a substrate-binding exosite offD. In some cases, any aptamer of the preceding blocks an active site offD. In some cases, any aptamer of the preceding blocks asubstrate-binding exosite of fD. In some cases, any aptamer of thepreceding blocks both an active site and a substrate-binding exosite offD. In some cases, any aptamer of the preceding inhibits a functionassociated with fD. In some cases, any aptamer of the preceding preventsassociation of fD with pre-formed C3bB complex. In some cases, anyaptamer of the preceding has no more than one nucleic acid strand. Inother cases, any aptamer of the preceding comprises more than onenucleic acid strand. In some cases, the nucleic acid sequence of anyaptamer of the preceding has from 30-90 nucleotides, non-nucleotidylspacers, or a combination thereof. In some cases, any aptamer of thepreceding selectively binds to an active site of fD with a K_(d) of lessthan about 50 nM. In some cases, any aptamer of the preceding inhibitsfD in an alternative complement dependent hemolysis assay with an IC₅₀of less than about 50 nM. In some cases, any aptamer of the precedinginhibits fD in a fD convertase assay with an IC₅₀ of less than about 50nM. In some cases, any aptamer of the preceding inhibits at least 85% offD activity in an alternative complement dependent hemolysis assay. Insome cases, any aptamer of the preceding inhibits at least 85% of fDactivity in a fD convertase assay. In some cases, any aptamer of thepreceding inhibits fD activity in an esterase activity assay. In somecases, any aptamer of the preceding binds to fD with a K_(d) of lessthan about 50 nM and inhibits fD in an alternative complement dependenthemolysis assay with an IC₉₀ of less than about 500 nM. In some cases,any aptamer of the preceding binds to fD with a K_(d) of less than about50 nM and inhibits fD in an alternative complement dependent hemolysisassay with an IC₅₀ of less than about 100 nM. In some cases, any aptamerof the preceding is conjugated to a polyethylene glycol (PEG) molecule.In some cases, the PEG molecule has a molecular weight of 80 kDa orless. In some cases, any aptamer of the preceding does not contain apseudoknot structure. In some cases, any aptamer of the preceding hasless than 3 unpaired nucleotide residues at a 5′ terminus, a 3′terminus, or both.

In another aspect, an aptamer is provided comprising a nucleic acidsequence comprising any one of SEQ ID NOs:13, 165, 166, 244, 253, 256,262, 269, 284, 285, 294, 303, 306, and 312, or comprising at least 80%sequence identity to any one of SEQ ID NOs:13, 165, 166, 244, 253, 256,262, 269, 284, 285, 294, 303, 306, and 312. In some cases, the nucleicacid sequence comprises one or more modified nucleotides. In someinstances, at least 50% of said nucleic acid sequence comprises the oneor more modified nucleotides. In some cases, the one or more modifiednucleotides comprises a 2′F-modified nucleotide, a 2′OMe-modifiednucleotide, or a combination thereof. In some cases, the one or moremodified nucleotides are selected from the group consisting of: 2′F-G,2′OMe-G, 2′OMe-U, 2′OMe-A, 2′OMe-C, a 3′ terminal inverteddeoxythymidine, and any combination thereof. In some cases, the aptameris selected from the group consisting of: Aptamer 76 as described inTable 2, Aptamer 116 as described in Table 2, Aptamer 102 as describedin Table 2, Aptamer 104 as described in Table 2, Aptamer 106 asdescribed in Table 2, Aptamer 108 as described in Table 2, Aptamer 107as described in Table 2, Aptamer 109 as described in Table 2, andAptamer 99 as described in Table 2. In some cases, the aptamer isconjugated to a polyethylene glycol (PEG) molecule. In some cases, thePEG molecule has a molecular weight of 80 kDa or less. In some cases,the PEG molecule is conjugated to the aptamer using a pegylationreagent, wherein the pegylation reagent comprises2,3-Bis(methylpolyoxyethylene-oxy)-1-{3-[(1,5-dioxo-5-succinimidyloxy,pentyl)amino]propyloxy} propane.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively blocks the active site of complement factor D(fD) and having a stem-loop secondary structure comprising at least onestem and at least one loop.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising at least one stem and at leastone loop, wherein the aptamer comprises at least one modifiednucleotide. In some cases, the aptamer comprises a nuclease-stabilizednucleic acid backbone.

In another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising at least one stem and at leastone loop, wherein the nucleic acid sequence does not include any one ofSEQ ID NOs:228-235.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively blocks an active site of complement factor D(fD) and having a secondary structure having exactly three loops. Insome cases, the secondary structure further has exactly two base-pairedstems.

In some cases, an aptamer of any of the preceding has a nucleic acidsequence that does not include any one of SEQ ID NOs:1-3, 168-227.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D and having astem-loop secondary structure comprising at least one stem and at leastone loop, wherein said aptamer is an RNA aptamer or a modified RNAaptamer.

In some cases, an aptamer of any of the preceding further comprises upto two stems. In some cases, an aptamer of any of the preceding furthercomprises up to three loops. In some cases, an aptamer of any of thepreceding is an RNA aptamer or a modified RNA aptamer. In some cases, anaptamer of any of the preceding is a DNA aptamer or a modified DNAaptamer. In some cases, an aptamer of any of the preceding selectivelybinds to an active site of fD. In some cases, an aptamer of thepreceding has at least one loop, wherein each of the at least one loophas up to 25 nucleotides. In some cases, an aptamer of any of thepreceding has no more than one nucleic acid strand. In some cases, anaptamer of any of the preceding has at least one stem, wherein no morethan one of the at least one stem has more than 20 base pairs. In somecases, an aptamer of any of the preceding has a nucleic acid sequencecomprising from 30-90 nucleotides.

In some cases, an aptamer of any of the preceding has a stem-loopsecondary structure comprising, in a 5′ to 3′ direction, a first stem, afirst loop, a second stem, a second loop, and a third loop. In somecases, the first loop comprises fewer nucleotides than the second loop.In some cases, the third loop is connected to the first stem. In somecases, the first loop has from 1 to 10 nucleotides. In some cases, thefirst loop has from 3 to 5 nucleotides. In some cases, the first loopcomprises a nucleic acid sequence of 5′-DUG-3′, where D is A, G, or U.In some cases, the second loop has from 2 to 15 nucleotides. In somecases, the second loop has at least 8 nucleotides. In some cases, thesecond loop has exactly 10 nucleotides. In some cases, the second loophas 10 or 11 nucleotides. In some cases, the second loop comprises anucleic acid sequence of 5′-DWWVGCBHWG-3′ (SEQ ID NO:319), where D is A,G, or U; W is A or U; V is A, C, or G; B is C, G, or U; and H is A, C,or U. In some cases, the second loop comprises a nucleic acid sequencehaving a U at nucleotide position 2, position 3, or both. In some cases,the third loop has from 2 to 10 nucleotides. In some cases, the thirdloop has at least 6 nucleotides. In some cases, the third loop hasexactly 6 nucleotides. In some cases, the third loop has from 6 to 8nucleotides. In some cases, the third loop has a nucleic acid sequencecomprising 5′-AAGUKN-3′, where K is G or U; and N is A, G, C, or U. Insome cases, the first stem has from 2 to 10 base pairs. In some cases,the first stem has from 3 to 8 base pairs. In some cases, the secondstem has from 2 to 10 base pairs. In some cases, the second stem has 4or 5 base pairs. In some cases, the second stem comprises a terminal U-Gbase pair adjacent to the second loop. In some cases, the second stemcomprises a terminal C-G base pair adjacent to the second loop.

In some cases, an aptamer of any of the preceding selectively binds toan active site of fD with a K_(d) of less than about 50 nM. In somecases, an aptamer of any of the preceding inhibits fD in an alternativecomplement dependent hemolysis assay with an IC₅₀ of less than about 50nM. In some cases, an aptamer of any of the preceding inhibits fD in afD convertase assay with an IC₅₀ of less than about 50 nM. In somecases, an aptamer of any of the preceding inhibits at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an aptamer of any of the preceding inhibits at least 85% of fDactivity in a fD convertase assay. In some cases, an aptamer of any ofthe preceding inhibits fD activity in an esterase activity assay. Insome cases, an aptamer of any of the preceding binds to fD with a IQ ofless than about 50 nM and inhibits fD in an alternative complementdependent hemolysis assay with an IC₉₀ of less than about 500 nM. Insome cases, an aptamer of any of the preceding binds to fD with a K_(d)of less than about 50 nM and inhibits fD in an alternative complementdependent hemolysis assay with an IC₅₀ of less than about 100 nM. Insome cases, an aptamer of any of the preceding has a nucleic acidsequence comprising at least one modified nucleotide. In some cases, anaptamer of any of the preceding is conjugated to a polyethylene glycol(PEG) molecule. In some cases, the PEG molecule has a molecular weightof 80 kDa or less.

In another aspect, an aptamer is provided having a nucleic acid sequencecomprising any one of SEQ ID NOs:1-3, 10-167, 267-286, 317, and 318, orany nucleic acid sequence described in Table 2 or having at least 80%sequence identity to any one of SEQ ID NOs:1-3, 10-167, 267-286, 317,and 318, or any nucleic acid sequence described in Table 2.

In yet another aspect, an aptamer is provided comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising a terminal stem, an asymmetricinternal loop, an internal stem, and a terminal loop.

In some cases, an aptamer of any of the preceding does not contain apseudoknot structure. In some cases, an aptamer of any of the precedinghas less than 3 unpaired nucleotide residues at a 5′ terminus, a 3′terminus, or both.

In another aspect, an aptamer according to any of the preceding isprovided for use in a method of therapy; for use in a method oftreatment that benefits from modulating fD; for use in a method oftreatment that benefits from inhibiting a function associated with fD;or for use in a method for the treatment of ocular diseases.

In another aspect, an aptamer according to any of the preceding isprovided and a pharmaceutically acceptable carrier, excipient, ordiluent. In some cases, a pharmaceutical composition or medicament isprovided comprising a plurality of aptamers according to any of thepreceding. In some cases, greater than 90% of the plurality of aptamerscomprise nucleotides having ribose in a β-D-ribofuranose configuration.

In yet another aspect, a method is provided for modulating complementfactor D (fD) in a biological system, the method comprising:administering to the biological system, an aptamer according to any oneof the preceding, thereby modulating fD in the biological system. Insome cases, the modulating comprises inhibiting a function associatedwith fD. In some cases, the modulating comprises preventing associationof fD with pre-formed C3bB complex. In some cases, the biological systemis a subject. In some cases, the subject is a human.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesto the same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts a non-limiting example of a consensus secondary structureof a family of stem-loop anti-fD aptamers according to an embodiment ofthe disclosure (SEQ ID NO:320). FIG. 1 further depicts a non-limitingillustration of numbering of stem and loop sequences according to anembodiment of the disclosure.

FIG. 2 depicts a non-limiting example of an asymmetric loop according toan embodiment of the disclosure (SEQ ID NO:321).

FIG. 3 depicts a non-limiting example of a role for the alternativecomplement pathway in the pathogenesis of geographic atrophy.

FIG. 4A and FIG. 4B depict modeling of the intravitreal (IVT) inhibitionof Factor D by an anti-Factor D aptamer at various IVT concentrationsover time.

FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D depict non-limiting examples ofsmall molecule inhibitors of fD.

FIG. 6 depicts the amino acid sequence of human complement Factor D,chymotrypsin numbering scheme, and fD numbering scheme.

FIG. 7A, FIG. 7B, and FIG. 7C depict a non-limiting example of anaptamer library sequence that may be utilized to generate anti-Factor Daptamers according to an embodiment of the disclosure (SEQ ID NOs: 322,323, and 6, in order of appearance).

FIG. 8 depicts binding analysis of libraries enriched in anti-Factor Daptamers by flow cytometry according to an embodiment of the disclosure.

FIG. 9 depicts measurement of K_(d) values of libraries enriched inanti-Factor D aptamers according to an embodiment of the disclosure.

FIG. 10 depicts direct binding analysis of anti-Factor D aptamers byflow cytometry according to an embodiment of the disclosure.

FIG. 11 depicts measurement of K_(d) values of anti-Factor D aptamersaccording to an embodiment of the disclosure.

FIG. 12 depicts a competition assay according to an embodiment of thedisclosure.

FIG. 13 depicts examples of data obtained from an alternative complementdependent hemolysis assay according to an embodiment of the disclosure.

FIG. 14 depicts examples of data obtained from a fD esterase activityassay according to an embodiment of the disclosure.

FIG. 15A depicts examples of data obtained from an alternativecomplement dependent hemolysis assay according to an embodiment of thedisclosure. FIG. 15B depicts examples of data obtained from a fDesterase activity assay according to an embodiment of the disclosure.

FIG. 15C depicts examples of data obtained from a competition assayaccording to an embodiment of the disclosure.

FIG. 16 depicts examples of data obtained from selective substitution of3-carbon spacers for each nucleotide of a fD aptamer according to anembodiment of the disclosure (SEQ ID NO:12).

FIG. 17 depicts examples of data obtained from an alternative complementdependent hemolysis assay according to an embodiment of the disclosure.

FIG. 18 depicts examples of data obtained from a competition bindingassay according to an embodiment of the disclosure.

FIG. 19A and FIG. 19B depict examples of data obtained from analternative complement dependent hemolysis assay according to anembodiment of the disclosure.

FIG. 20A, FIG. 20B, FIG. 20C, and FIG. 20D depict non-limiting examplesof secondary structures of several active-site directed inhibitors of fDaccording to an embodiment of the disclosure (SEQ ID NOs: 162, and165-167, in order of appearance).

FIG. 21 depicts examples of relative binding affinity of severalactive-site directed inhibitors of fD using a flow cytometry basedcompetition binding assay according to an embodiment of the disclosure.

FIG. 22 depicts a non-limiting example of SPR complex assembly dataaccording to an embodiment of the disclosure.

FIG. 23 depicts a non-limiting example of dose-dependent inhibition ofC3bB:inactivated fD complex assembly with a fD aptamer according to anembodiment of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure herein provides aptamer compositions that selectivelybind to and inhibit a function associated with complement factor D (fD)and methods of using such aptamer compositions. Specifically, theaptamer compositions described herein have unique stem-loop secondarystructures. In some cases, the aptamers of the disclosure have, in a 5′to 3′ direction, a first base paired stem, a first loop, a second basepaired stem, a second loop, and a third loop. The aptamers may alsoinclude one or more further elements (e.g., additional stem(s) orloop(s)). In some cases, such further elements are located before thefirst base paired stem and/or after the third loop. In some cases, suchfurther elements are located interspersed between other elements of theaptamer (e.g., between the first loop and the second base paired stem,etc.). In other embodiments, each element is adjacent to each other. Forexample, the aptamers may have, in a 5′ to 3′ direction, a first basepaired stem adjacent to a first loop, which is adjacent to a second basepaired stem, which is adjacent to a second loop. A third loop may bepresent, and may, in some cases be adjacent to the first and/or secondbase paired stems. In some cases, the aptamers of the disclosure have aterminal base paired stem, an asymmetric internal loop, an internal basepaired stem, and/or a terminal loop. Non-limiting examples of stem-loopaptamers that may be used to inhibit fD are described throughout.

The disclosure herein provides methods and compositions for thetreatment of ocular diseases or disorders. In some cases, the methodsand compositions include the use of an anti-fD stem-loop aptamer for,e.g., the treatment of ocular diseases or disorders. In some cases, theocular disease is macular degeneration. In some cases, maculardegeneration is age-related macular degeneration. In some cases,age-related macular degeneration is dry age-related maculardegeneration. In some cases, dry age-related macular degeneration isadvanced dry age-related macular degeneration (i.e., geographicatrophy). In some cases, the ocular disease is wet age-related maculardegeneration. In some cases, the ocular disease is Stargardt disease. Insome cases, the methods and compositions involve the inhibition of thealternative complement pathway. In some cases, the methods andcompositions involve the inhibition of a function associated with FactorD (fD). In some cases, the methods and compositions involve theinhibition of a function associated with fD for the treatment of oculardiseases. In some cases, the methods and compositions involve theinhibition of a function associated with fD for the treatment of dryage-related macular degeneration or geographic atrophy. In some cases,the methods and compositions involve the inhibition of a functionassociated with fD for the treatment of wet age-related maculardegeneration. In some cases, the methods and compositions involve theinhibition of a function associated with fD for the treatment ofStargardt disease.

In various aspects, the compositions may include oligonucleotides (e.g.,aptamers) that selectively bind to and modulate an activity associatedwith fD. In some instances, the oligonucleotide compositions of thedisclosure inhibit a function associated with fD. In some cases, theoligonucleotide compositions may bind directly to an active site of fDor to a region of fD that includes the active site, or theoligonucleotide compositions may bind to a region of fD such that theoligonucleotide occludes or blocks access to the active site. In somecases, the oligonucleotide compositions may bind directly to an exositeof fD or to a region of fD that includes the exosite, or theoligonucleotide compositions may bind to a region of fD such that theoligonucleotide occludes or blocks access of a substrate to the exosite.In some cases, the oligonucleotide compositions may bind to and/or blockaccess to both the active site and the exosite of fD. In some cases, theoligonucleotide compositions may bind to the active site of fD and blockaccess to the exosite of fD. In some cases, the oligonucleotidecompositions may block access to the active site of fD and bind to theexosite of fD. In some cases, the oligonucleotides are aptamers, such asRNA aptamers, DNA aptamers, modified RNA aptamers, or modified DNAaptamers. In particular examples, the aptamers of the disclosure mayhave secondary structures. The secondary structures may include astem-loop structure which may include one or more loops and one or morestems. Various examples of aptamers having stem-loop structures formodulating fD are described herein.

The practice of some embodiments disclosed herein employ, unlessotherwise indicated, conventional techniques of immunology,biochemistry, chemistry, molecular biology, microbiology, cell biology,genomics and recombinant DNA, which are within the skill of the art. Seefor example Sambrook and Green, Molecular Cloning: A Laboratory Manual,4th Edition (2012); the series Current Protocols in Molecular Biology(F. M. Ausubel, et al. eds.); the series Methods In Enzymology (AcademicPress, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hamesand G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies,A Laboratory Manual, and Culture of Animal Cells: A Manual of BasicTechnique and Specialized Applications, 6th Edition (R. I. Freshney, ed.(2010)).

In general, “sequence identity” refers to an exactnucleotide-to-nucleotide or amino acid-to-amino acid correspondence oftwo polynucleotides or polypeptide sequences, respectively. Typically,techniques for determining sequence identity include determining thenucleotide sequence of a polynucleotide and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Two or more sequences (polynucleotideor amino acid) can be compared by determining their “percent identity.”The percent identity of two sequences, whether nucleic acid or aminoacid sequences, is the number of exact matches between two alignedsequences divided by the length of the longer sequences and multipliedby 100. Percent identity may also be determined, for example, bycomparing sequence information using the advanced BLAST computerprogram, including version 2.2.9, available from the National Institutesof Health. The BLAST program is based on the alignment method of Karlinand Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and asdiscussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); KarlinAnd Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); andAltschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, theBLAST program defines identity as the number of identical alignedsymbols (generally nucleotides or amino acids), divided by the totalnumber of symbols in the shorter of the two sequences. The program maybe used to determine percent identity over the entire length of theproteins being compared. Default parameters are provided to optimizesearches with short query sequences in, for example, with the blastpprogram. The program also allows use of an SEG filter to mask-offsegments of the query sequences as determined by the SEG program ofWootton and Federhen, Computers and Chemistry 17:149-163 (1993). Rangesof desired degrees of sequence identity are approximately 80% to 100%and integer values therebetween. Typically, the percent identitiesbetween a disclosed sequence and a claimed sequence are at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98%.

The term “aptamer” as used herein refers to an oligonucleotide and/ornucleic acid analogues that can bind to a specific target molecule.Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleicacid analogue, and/or combinations thereof. Aptamers can besingle-stranded oligonucleotides. In some cases, aptamers may comprisemore than one nucleic acid strand (e.g., two or more nucleic acidstrands). Without wishing to be bound by theory, aptamers are thought tobind to a three-dimensional structure of a target molecule. Aptamers maybe monomeric (composed of a single unit) or multimeric (composed ofmultiple units). Multimeric aptamers can be homomeric (composed ofmultiple identical units) or heteromeric (composed of multiplenon-identical units). Aptamers herein may be described by their primarystructures, meaning the linear nucleotide sequence of the aptamer.Aptamer sequences herein are generally described from the 5′ end to the3′ end, unless otherwise stated. Additionally or alternatively, aptamersherein may be described by their secondary structures which may refer tothe combination of single-stranded regions and base-pairing interactionswithin the aptamer.

An aptamer may have a secondary structure having at least twocomplementary regions of the same nucleic acid strand that base-pair toform a double helix (referred to herein as a “stem”). Generally, thesecomplementary regions are complementary when read in the oppositedirection. The term “stem” as used herein may refer to either of thecomplementary nucleotide regions individually or may encompass abase-paired region containing both complementary regions, or a portionthereof. For example, the term “stem” may refer to the 5′ side of thestem, that is, the stem sequence that is closer to the 5′ end of theaptamer; additionally or alternatively, the term “stem” may refer to the3′ side of the stem, that is, the stem sequence that is closer to the 3′end of the aptamer. In some cases, the term “stem” may refer to the 5′side of the stem and the 3′ side of the stem, collectively. The term“base-paired stem” is generally used herein to refer to bothcomplementary stem regions collectively. A base-paired stem may beperfectly complementary meaning that 100% of its base pairs areWatson-Crick base pairs. A base-paired stem may also be “partiallycomplementary.” As used herein, the term “partially complementary stem”refers to a base-paired stem that is not entirely made up ofWatson-Crick base pairs but does contain base pairs (either Watson-Crickbase pairs or G-U/U-G wobble base pairs) at each terminus. In somecases, a partially complementary stem contains both Watson-Crickbase-pairs and G-U/U-G wobble base pairs. In other cases, a partiallycomplementary stem is exclusively made up of G-U/U-G wobble base pairs.A partially complementary stem may contain mis-matched base pairs and/orunpaired bases in the region between the base pairs at each terminus ofthe stem; but in such cases, the mis-matched base pairs and/or unpairedbases make up at most 50% of the positions between the base pairs ateach terminus of the stem.

A stem as described herein may be referred to by the position, in a 5′to 3′ direction on the aptamer, of the 5′ side of the stem (i.e., thestem sequence closer to the 5′ terminus of the aptamer), relative to the5′ side of additional stems present on the aptamer. For example, asdepicted in FIG. 1, stem 1 (S1) may refer to the stem sequence that isclosest to the 5′ terminus of the aptamer, its complementary stemsequence, or both stem sequences collectively. Similarly, stem 2 (S2)may refer to the next stem sequence that is positioned 3′ relative toS1, its complementary stem sequence, or both stem sequencescollectively. In some cases, the aptamers of the disclosure have exactlytwo stems (e.g., S1 and S2). In other cases, the aptamers of thedisclosure may have more than two stems (e.g., S1, S2, S3, etc.). Eachadditional stem may be referred to by its position, in a 5′ to 3′direction, on the aptamer, as described above. For example, S3 may bepositioned 3′ relative to S2 on the aptamer, S4 may be positioned 3′relative to S3 on the aptamer, and so on. In some cases, the term “firststem” is used to refer to a stem in the aptamer, irrespective of itslocation. For example, a first stem may be S1, S2, S3, S4 or any otherstem in the aptamer.

A stem may be adjacent to an unpaired region. An unpaired region may bepresent at a terminus of the aptamer or at an internal region of theaptamer.

As used herein, the term “loop” generally refers to an internal unpairedregion of an aptamer. The term “loop” generally refers to any unpairedregion of an aptamer that is flanked on both the 5′ end and the 3′ endby a stem region. In some cases, a loop sequence may be adjacent to asingle base-paired stem, such that the loop and stem structure togetherresemble a hairpin. In such cases, generally the primary sequence of theaptamer contains a first stem sequence adjacent to the 5′ end of theloop sequence and a second stem sequence adjacent to the 3′ end of theloop sequence; and the first and second stem sequences are complementaryto each other. In some cases, each terminus of a loop is adjacent tofirst and second stem sequences that are not complementary. In suchcases, the primary sequence of the aptamer may contain an additionalloop sequence that is bordered at one or both ends by stem sequencesthat are complementary to the first and/or second stem sequences. Incases where the two loops have different number of nucleotides, the twoloops are referred to jointly herein as an “asymmetric loop” or“asymmetric loop pair,” terms that are used herein interchangeably. Incases where the two loops have the same number of nucleotides, they arereferred to jointly as a “symmetric loop” or “symmetric loop pair,”terms that are used interchangeably herein. FIG. 2 depicts an example ofan “asymmetric loop”, composed of two loops that each contain differentnumbers of nucleotides and that border the same two stems. In thisexample, the first loop sequence has 3 nucleotides, and the second loopsequence has 6 nucleotides. An “asymmetric loop” is bordered by exactlytwo base-paired stems, as depicted in the example shown in FIG. 2.Similarly, a “symmetric loop” is bordered by exactly two base-pairedstems.

A loop as described herein may be referred to by its position, in a 5′to 3′ direction, on the aptamer. For example, as depicted in FIG. 1,loop 1 (L1) may refer to a loop sequence that is positioned most 5′ onthe aptamer. Similarly, loop 2 (L2) may refer to a loop sequence that ispositioned 3′ relative to L1, and loop 3 (L3) may refer to a loopsequence that is positioned 3′ relative to L2. In some cases, theaptamers of the disclosure have exactly three loops (e.g., L1, L2, andL3). In other cases, the aptamers of the disclosure may have more thanthree loops (e.g., L1, L2, L3, L4, etc.). Each additional loop may bereferred to by its position, in a 5′ to 3′ direction, on the aptamer, asdescribed above. For example, L4 may be positioned 3′ relative to L3 onthe aptamer, L5 may be positioned 3′ relative to L4 on the aptamer, andso on. In some cases, the term “first loop” is used to refer to a loopin the aptamer, irrespective of its location. For example, a first loopmay be L1, L2, L3, L4 or any other loop in the aptamer.

The term “stem-loop” as used herein generally refers to the secondarystructure of an aptamer of the disclosure having at least one stem andat least one loop. In some cases, a stem-loop secondary structure mayinclude a terminal stem and a terminal loop. In some cases, a stem-loopsecondary structure includes structures having two stems, which mayinclude a terminal stem, an internal loop, an internal stem, and aterminal loop. A “terminal stem” as used herein generally refers to astem that encompasses both the 5′ and/or 3′ terminus of the aptamer. Insome cases, a “terminal stem” is bordered at one or both termini by a“tail” comprising one or more unpaired nucleotides. For example, aterminal stem present in the aptamer may be bordered by a tail of one ormore unpaired nucleotides (or other structures) at its 5′ end.Similarly, a terminal stem present in the aptamer may be bordered by atail of one or more unpaired nucleotides (or other structures) at its 3′end. In some cases, a terminal stem present in the aptamer may bebordered by a tail of one or more unpaired nucleotides (or otherstructures) at both its 5′ end and its 3′ end. A terminal stem isgenerally adjacent to a loop; for example, the 5′ side of a terminalstem (i.e., the terminal stem sequence closest to the 5′ end of themolecule) may be bordered at its 3′ terminus by the 5′ terminus of aloop. Similarly, the 3′ side of a terminal stem (i.e., the terminal stemsequence closest to the 3′ end of the molecule) may be bordered at its5′ terminus by the 3′ terminus of a loop. An “internal stem” as usedherein generally refers to a stem that is bordered at both termini by aloop sequence. A “terminal loop” as used herein generally refers to aloop that is bordered by the same stem at both termini of the loop. Forexample, a terminal loop may be bordered at its 5′ end by a stemsequence, and may be bordered at its 3′ end by the complementary stemsequence. An “internal loop” as used herein generally refers to a loopthat is bordered at both termini by different stems. For example, aninternal loop may be bordered at its 5′ end by a first stem sequence,and may be bordered at its 3′ end by a second stem sequence that is notcomplementary to the first stem sequence. In some cases, a stem-loopsecondary structure includes structures having more than two stems.Unless otherwise stated, when an aptamer includes more than one stemand/or more than one loop, the stems and loops are numberedconsecutively in ascending order from the 5′ end to the 3′ end of theprimary nucleotide sequence.

In some cases, an aptamer of the disclosure may have a terminal stem, anasymmetric internal loop, an internal stem, and a terminal loop, such asdepicted in FIG. 1. In some cases, an aptamer of the disclosure may haveexactly one terminal stem, exactly one asymmetric internal loop, exactlyone internal stem, and exactly one terminal loop. In some cases, anaptamer of the disclosure may have, in a 5′ to 3′ direction, a firststem, a first loop, a second stem, a second loop, and a third loop. Insome cases, an aptamer of the disclosure may have the general structure,in a 5′ to 3′ direction, S1-L1-S2-L2-S2-L3-S1 (FIG. 1).

The term “exosite” as used herein generally refers to a protein domainor region of a protein that is capable of binding to another protein.The exosite may also be referred to herein as a “secondary bindingsite”, for example, a binding site that is remote from or separate froma primary binding site (e.g., an active site). In some cases, theprimary and secondary binding sites may overlap. Binding of a moleculeto an exosite may cause a physical change in the protein (e.g., aconformational change). In some cases, the activity of a protein may bedependent on occupation of the exosite. In some examples, the exositemay be distinct from an allosteric site. In some cases, theoligonucleotide compositions of the disclosure may bind to the exositeof fD or to part of the exosite of fD, or may bind to a region of fDthat includes the exosite. In some cases, the oligonucleotidecompositions of the disclosure may block or occlude the exosite suchthat the natural substrate of fD is prevented from accessing theexosite. In such cases, the oligonucleotide may block access to theexosite without directly binding the exosite (e.g., may bind to a regionof fD other than the exosite in such a way that the exosite issterically occluded).

The term “catalytic cleft” or “active site” as used herein refers to adomain of an enzyme in which a substrate molecule binds to and undergoesa chemical reaction. The active site may include amino acid residuesthat form temporary bonds with the substrate (e.g., a binding site) andamino acid residues that catalyze a reaction of that substrate (e.g.,catalytic site). The active site may be a groove or pocket (e.g., acleft) of the enzyme which can be located in a deep tunnel within theenzyme or between the interfaces of multimeric enzymes. In some cases,the oligonucleotide compositions of the disclosure may bind to theactive site of fD or to part of the active site of fD, or may bind to aregion of fD that includes the active site. In some cases, theoligonucleotide compositions of the disclosure may block or occlude theactive site of fD such that the natural substrate of fD is preventedfrom accessing the active site. In such cases, the oligonucleotide mayblock access to the active site, without directly binding the activesite (e.g., may bind to a region of fD other than the active site insuch a way that the active site is sterically occluded). In some cases,the oligonucleotide compositions of the disclosure may includeoligonucleotides that block or occlude the active site of fD, withoutdirectly binding the constituent amino acids comprising the active siteof fD, such that the natural substrate of fD is prevented from accessingthe active site.

In some cases, oligonucleotide compositions (e.g., aptamers) of thedisclosure may block or occlude both the active site and the exosite.For example, oligonucleotide compositions (e.g., aptamers) of thedisclosure may both block access to the active site and may block accessto the substrate-binding exosite. In some cases, oligonucleotidecompositions of the disclosure may bind to and/or block access to theactive site of fD and prevent association of fD with pre-formed C3bBcomplex. In some cases, oligonucleotide compositions of the disclosuremay bind to and/or block access to both the active site and thesubstrate-binding exosite of fD, and may prevent association of fD withpre-formed C3bB complex.

The term “epitope” as used herein refers to the part of an antigen(e.g., a substance that stimulates an immune system to generate anantibody against) that is specifically recognized by the antibody. Insome cases, the antigen is a protein or peptide and the epitope is aspecific region of the protein or peptide that is recognized and boundby an antibody. In some cases, the aptamers described herein bind to aregion of fD that is an epitope for an anti-fD antibody or antibodyfragment thereof, wherein the anti-fD antibody inhibits a functionassociated with fD. In some cases, the aptamer binding region of fDoverlaps with at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or 100% of the epitope for an anti-fD antibodyor the binding site of another fD-inhibiting molecule.

The terms “peptide” and “protein” are used interchangeably herein torefer to polymers of amino acids of any length. A polypeptide can be anyprotein, peptide, protein fragment or component thereof. A polypeptidecan be a protein naturally occurring in nature or a protein that isordinarily not found in nature. A polypeptide can consist largely of thestandard twenty protein-building amino acids or it can be modified toincorporate non-standard amino acids. A polypeptide can be modified,typically by the host cell, by e.g., adding any number of biochemicalfunctional groups, including phosphorylation, acetylation, acylation,formylation, alkylation, methylation, lipid addition (e.g.palmitoylation, myristoylation, prenylation, etc) and carbohydrateaddition (e.g. N-linked and O-linked glycosylation, etc). Polypeptidescan undergo structural changes in the host cell such as the formation ofdisulfide bridges or proteolytic cleavage. The peptides described hereinmay be therapeutic peptides utilized for e.g., the treatment of adisease.

The Complement System and the Alternative Complement Pathway

The complement system is a part of the innate immune system thatenhances the ability of antibodies and phagocytic cells to clearpathogens from an organism. Although the system is not adaptable anddoes not change over the course of an individual's lifetime, it can berecruited and brought into action by the adaptive immune system.

The complement system consists of a number of small proteins found inthe blood, in general synthesized by the liver, and normally circulatingas inactive precursors (pro-proteins). When stimulated by one of severaltriggers, proteases in the system cleave specific proteins to releasecytokines and initiate an amplifying cascade of further cleavages. Theend result of this complement activation or complement fixation cascadeis massive amplification of the response and activation of thecell-killing membrane attack complex. Over 30 proteins and proteinfragments make up the complement system, including serum proteins,serosal proteins, and cell membrane receptors.

The alternative complement pathway is a rapid, antibody-independentroute for complement system activation and amplification. Thealternative pathway comprises several components: C3, Factor B (fB), andfD. Activation of the alternative pathway occurs when C3b, a proteolyticcleavage form of C3, is bound to an activating surface agent such as abacterium. fB is then bound to C3b, and cleaved by fD to yield the C3convertase C3bBb. Amplification of C3 convertase activity occurs asadditional C3b is produced and deposited. The amplification response isfurther aided by the binding of the positive regulator protein properdin(Factor P), which stabilizes the active convertase against degradation,extending its half-life from 1-2 minutes to 18 minutes.

The C3 convertase further assembles into a C5 convertase (C3b3bBb). Thiscomplex subsequently cleaves complement component C5 into twocomponents: the C5a polypeptide (9 kDa) and the C5b polypeptide (170kDa). The C5a polypeptide binds to a 7 transmembrane G-protein coupledreceptor, which was originally associated with leukocytes and is nowknown to be expressed on a variety of tissues including hepatocytes andneurons. The C5a molecule is the primary chemotactic component of thehuman complement system and can trigger a variety of biologicalresponses including leukocyte chemotaxis, smooth muscle contraction,activation of intracellular signal transduction pathways,neutrophil-endothelial adhesion, cytokine and lipid mediator release andoxidant formation.

The alternative complement pathway is believed to play a role in thepathogenesis of a variety of ischemic, inflammatory and autoimmunediseases including age-related macular degeneration, geographic atrophy,Stargardt disease, systemic lupus erythematosus, rheumatoid arthritis,and asthma. Thus, components of the alternative complement pathway maybe important targets for the treatment of these diseases.

Age-Related Macular Degeneration

Age-related macular degeneration (“AMD”) is a chronic and progressiveeye disease that is the leading cause of irreparable vision loss in theUnited States, Europe, and Japan. AMD is characterized by theprogressive deterioration of the central portion of the retina referredto as the macula. The clearest indicator of progression to AMD is theappearance of drusen, yellow-white deposits under the retina, which areplaques of material that are derived from the metabolic waste productsof retinal cells. The appearance of drusen is an important component ofboth forms of AMD: exudative (“wet”) and non-exudative (“dry”). Thepresence of numerous, intermediate-to-large drusen is associated withthe greatest risk of progression to late-stage disease, characterized bygeographic atrophy and/or neovascularization. The majority of patientswith wet AMD experience severe vision loss in the affected eye withinmonths to two years after diagnosis of the disease, although vision losscan occur within hours or days. Dry AMD is more gradual and occurs whenlight-sensitive cells in the macula slowly atrophy, gradually blurringcentral vision in the affected eye. Vision loss is exacerbated by theformation and accumulation of drusen and sometimes the deterioration ofthe retina, although without abnormal blood vessel growth and bleeding.Geographic atrophy is a term used to refer to advanced dry AMD.Geographic atrophy is characterized by an “island” of atrophiedphotoreceptors cells. It is believed that the alternative complementpathway may play a role in the pathogenesis of AMD.

For example, FIG. 3 depicts a potential role for the alternativecomplement pathway in the pathogenesis of geographic atrophy. In thisexample, multiple factors may lead to activation of the alternativecomplement pathway, including the appearance of drusen in the eye,immune dysfunction, and genetic differences that predispose patients toactivation of the complement pathway. As described above, amplificationof C3 convertase activity may occur as additional C3b is produced anddeposited. C3 convertase activity may lead to inflammation andopsonization. The C3 convertase may further assemble into a C5convertase (C3b3bBb) which may lead to cell death through formation ofthe Membrane Attack Complex.

In some aspects, the oligonucleotide compositions of the disclosure maybe used to treat AMD. In some cases, the oligonucleotide compositions ofthe disclosure may be used to treat wet AMD. In some cases, theoligonucleotide compositions of the disclosure may be used to treatgeographic atrophy. In some cases, the oligonucleotide compositions ofthe disclosure may be used to stop, slow, or reverse the progression ofwet AMD or geographic atrophy. In some cases, the oligonucleotidecompositions of the disclosure may be used to treat symptoms associatedwith wet AMD or geographic atrophy.

Stargardt Disease

Stargardt Disease (“STGD”) is a rare, genetic, macular dystrophy with anincidence of 1/20,000, affecting approximately 30,000 individuals in theUnited States. STGD is an autosomal recessive or complex heterozygousgenetic disease caused by mutations in the ABCA4 gene. The ABCA4 geneencodes the photoreceptor protein ABCA4 Transporter, which isresponsible for removal of bisretinoid fluorophores, which can includeN-retinylidene-N-retinyethanolamine (A2E), all-trans-retinal and relatedphoto-oxidation products of vitamin A aldehyde which together constitutelipofuscin from photoreceptor cells. Accumulation of all-trans-retinalin photoreceptor cells is believed to damage RPE cells via oxidativestress, and trigger or promote complement-mediated damage to RPE cells,leading to retinal atrophy. STGD is characterized by the progressivedeterioration of the central portion of the retina referred to as themacula, generally beginning in the first two decades of life. Theclearest indicator of progression of STGD is the appearance of drusen,yellow-white deposits under the retina, which are plaques of materialthat are derived from the metabolic waste products of retinal cells,including all-trans-retinal and other vitamin A-related metabolites. Theonset of STGD is typically between the ages of 6-20 years, with earlysymptoms including difficulties in reading and adjusting to light.Patients with childhood-onset STGD tend to develop early severe visualacuity loss, significantly compromised retinal function, and rapidretinal pigment epithelial (RPE) cell atrophy with accompanying loss ofretinal function. The median ages of onset and the median age atbaseline examination are 8.5 (range, 3-16) and 12 years (range, 7-16),respectively. Patients with adult-onset disease are more likely topreserve visual acuity for a longer time and show slighter retinaldysfunction. Accumulation of all-trans-retinal in photoreceptor cellsleads to inflammation, oxidative stress, deposition of auto-fluorescentlipofuscin pigments in the retinal pigment epithelium and retinalatrophy. Lipofuscin deposits (drusen), and oxidative products, triggerthe alternative complement pathway into an inflammatory response leadingto cell death. Data supporting the role of alternative complement inSTGD include human cell models, genetic mouse models and theaccumulation of complement factors in humans in drusen during diseaseprogression. Therefore, inhibitors of complement, particularlycomplement factor D, are anticipated to stop or slow the progression ofvision loss in individuals with STGD. A related disease termedStargardt-like macular dystrophy, also known as STGD3, is inherited in adominant autosomal manner and is due to mutations in the ELOVL4 gene.ELOVL4 encodes the ELOVL4 protein, ELOVL fatty acid elongase 4.Mutations in ELOVL4 protein associated with STGD lead to mis-folding andaccumulation of ELOVL4 protein aggregates in retinal cells, which impactretinal cell function, eventually leading to cell death and retinalatrophy. Complement pathway activation is also thought to play a role inStargardt-like disease, and therefore inhibitors of complement,particularly complement factor D, are anticipated to stop or slow theprogression of vision loss in individuals with Stargardt-like disease.

In some aspects, the oligonucleotide compositions of the disclosure maybe used to treat Stargardt and Stargardt-like disease. In some cases,the oligonucleotide compositions of the disclosure may be used to stop,slow, or reverse the progression of Stargardt and Stargardt-likedisease. In some cases, the oligonucleotide compositions of thedisclosure may be used to treat symptoms associated with Stargardt andStargardt-like disease.

Aptamers

In some cases, the methods and compositions described herein use one ormore aptamers for the treatment of an ocular disease. In some cases, themethods and compositions described herein utilize one or more aptamersfor modulating an activity associated with fD. The term aptamer as usedherein refers to oligonucleotide molecules that bind to a target (e.g.,a protein) with high affinity and specificity through non-Watson-Crickbase pairing interactions. Generally, the aptamers described herein arenon-naturally occurring oligonucleotides (i.e., synthetically produced)that are isolated and used for the treatment of a disorder or a disease.Aptamers can bind to essentially any target molecule including, withoutlimitation, proteins, oligonucleotides, carbohydrates, lipids, smallmolecules, and even bacterial cells. The aptamers described herein areoligonucleotides that bind to proteins of the alternative complementpathway. Whereas many naturally occurring oligonucleotides, such asmRNA, encode information in their linear base sequences, aptamersgenerally do not encode information in their linear base sequences.Further, aptamers can be distinguished from naturally occurringoligonucleotides in that binding of aptamers to target molecules isdependent upon secondary and tertiary structures of the aptamer.

Aptamers may be suitable as therapeutic agents and may be preferable toother therapeutic agents because: 1) aptamers may be fast and economicalto produce because aptamers can be developed entirely by in vitroprocesses; 2) aptamers may have low toxicity and may lack an immunogenicresponse; 3) aptamers may have high specificity and affinity for theirtargets; 4) aptamers may have good solubility; 5) aptamers may havetunable pharmacokinetic properties; 6) aptamers may be amenable tosite-specific conjugation of PEG and other carriers; and 7) aptamers maybe stable at ambient temperatures.

Aptamers as described herein may include any number of modificationsthat can affect the function or affinity of the aptamer. For example,aptamers may be unmodified or they may contain modified nucleotides toimprove stability, nuclease resistance or delivery characteristics.Examples of such modifications may include chemical substitutions at thesugar and/or phosphate and/or base positions, for example, at the 2′position of ribose, the 5 position of pyrimidines, and the 8 position ofpurines, various 2′-modified pyrimidines and modifications with 2′-amino(2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe) substituents. Insome cases, aptamers described herein comprise a 2′-OMe and/or a 2′Fmodification to increase in vivo stability. In some cases, the aptamersdescribed herein contain modified nucleotides to improve the affinityand specificity of the aptamers for a specific epitope, exosite oractive site. Examples of modified nucleotides include those modifiedwith guanidine, indole, amine, phenol, hydroxymethyl, or boronic acid.In other cases, pyrimidine nucleotide triphosphate analogs orCE-phosphoramidites may be modified at the 5 position to generate, forexample, 5-benzylaminocarbonyl-2′-deoxyuridine (BndU);5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxyuridine (PPdU);5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU);5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU);5-(N-(1-naphthylmethyl)carboxamide)-2′-deoxyuridine (NapdU);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU);5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU);5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-isobutylaminocarbonyl-2′-deoxyuridine (IbdU);5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU);5-(N-isobutylaminocarbonyl-2′-deoxyuridine (iBudU);5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU),5-(N-imidizolylethylcarboxyamide)-2′-deoxyuridine (ImdU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium)propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxyamide)-2′-O-methy luridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),5-(N-2-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-2-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-1-naphthylethylcarboxyamide)-2′-O-methyluridine,5-(N-1-naphthylethylcarboxyamide)-2′-fluorouridine,5-(N-2-naphthylethylcarboxyamide)-2′-O-methyluridine,5-(N-2-naphthylethylcarboxyamide)-2′-fluorouridine,5-(N-3-benzofuranylethylcarboxyamide)-2′-deoxyuridine (BFdU),5-(N-3-benzofuranylethylcarboxyamide)-2′-O-methyluridine,5-(N-3-benzofuranylethylcarboxyamide)-2′-fluorouridine,5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU),5-(N-3-benzothiophenylethylcarboxyamide)-2′-O-methyluridine,5-(N-3-benzothiophenylethylcarboxyamide)-2′-fluorouridine;5-[N-(1-morpholino-2-ethyl)carboxamide]-2′-deoxyuridine (MOEdu);R-tetrahydrofiranylmethyl-2′-deoxyuridine (RTMdU);3-methoxybenzyl-2′-deoxyuridine (3MBndU);4-methoxybenzyl-2′-deoxyuridine (4MBndU);3,4-dimethoxybenzyl-2′-deoxyuridine (3,4DMBndU);S-tetrahydrofuranylmethyl-2′-deoxyuridine (STMdU);3,4-methylenedioxyphenyl-2-ethyl-2′-deoxyuridine (MPEdU);4-pyridinylmethyl-2′-deoxyuridine (PyrdU); or1-benzimidazol-2-ethyl-2′-deoxyuridine (BidU);5-(amino-1-propenyl)-2′-deoxyuridine;5-(indole-3-acetamido-1-propenyl)-2′-deoxyuridine; or5-(4-pivaloylbenzamido-1-propenyl)-2′-deoxyuridine.

Modifications of the aptamers contemplated in this disclosure include,without limitation, those which provide other chemical groups thatincorporate additional charge, polarizability, hydrophobicity, hydrogenbonding, electrostatic interaction, and functionality to the nucleicacid aptamer bases or to the nucleic acid aptamer as a whole.Modifications to generate oligonucleotide populations that are resistantto nucleases can also include one or more substitute internucleotidelinkages, altered sugars, altered bases, or combinations thereof. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, phosphorothioate, phosphorodithioate, or alkyl phosphatemodifications, methylations, and unusual base-pairing combinations suchas the isobases isocytidine and isoguanosine. Modifications can alsoinclude 3′ and 5′ modifications such as capping, e.g., addition of a3′-3′-dT cap to increase exonuclease resistance.

Aptamers of the disclosure may generally comprise nucleotides havingribose in the β-D-ribofuranose configuration. In some cases, 100% of thenucleotides present in the aptamer have ribose in the β-D-ribofuranoseconfiguration. In some cases, at least 50% of the nucleotides present inthe aptamer have ribose in the β-D-ribofuranose configuration. In somecases, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% ofthe nucleotides present in the aptamer have ribose in theβ-D-ribofuranose configuration.

The length of the aptamer can be variable. In some cases, the length ofthe aptamer is less than 100 nucleotides. In some cases, the length ofthe aptamer is greater than 10 nucleotides. In some cases, the length ofthe aptamer is between 10 and 90 nucleotides. The aptamer can be,without limitation, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, or about 90 nucleotides inlength.

In some instances, a polyethylene glycol (PEG) polymer chain iscovalently bound to the aptamer, referred to herein as PEGylation.Without wishing to be bound by theory, PEGylation may increase thehalf-life and stability of the aptamer in physiological conditions. Insome cases, the PEG polymer is covalently bound to the 5′ end of theaptamer. In some cases, the PEG polymer is covalently bound to the 3′end of the aptamer. In some cases, the PEG polymer is covalently boundto specific site on a nucleobase within the aptamer, including the5-position of a pyrimidine or 8-position of a purine. In some cases, thePEG polymer is covalently bound to an abasic site within the aptamer.

In some cases, an aptamer described herein may be conjugated to a PEGhaving the general formula, H—(O—CH₂—CH₂)_(n)—OH. In some cases, anaptamer described herein may be conjugated to a methoxy-PEG (mPEG) ofthe general formula, CH₃O—(CH₂—CH₂—O)_(n)—H. In some cases, the aptameris conjugated to a linear chain PEG or mPEG. The linear chain PEG ormPEG may have an average molecular weight of up to about 30 kD. Multiplelinear chain PEGs or mPEGs can be linked to a common reactive group toform multi-arm or branched PEGs or mPEGs. For example, more than one PEGor mPEG can be linked together through an amino acid linker (e.g.,lysine) or another linker, such as glycerine. In some cases, the aptameris conjugated to a branched PEG or branched mPEG. Branched PEGs or mPEGsmay be referred to by their total mass (e.g., two linked 20 kD mPEGshave a total molecular weight of 40 kD). Branched PEGs or mPEGs may havemore than two arms. Multi-arm branched PEGs or mPEGs may be referred toby their total mass (e.g. four linked 10 kD mPEGs have a total molecularweight of 40 kD). In some cases, an aptamer of the present disclosure isconjugated to a PEG polymer having a total molecular weight from about 5kD to about 200 kD, for example, about 5 about 10 kD, about 20 kD, about30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, about 80 kD,about 90 kD, about 100 kD, about 110 kD, about 120 kD, about 130 kD,about 140 kD, about 150 kD, about 160 kD, about 170 kD, about 180 kD,about 190 kD, or about 200 kD. In one non-limiting example, the aptameris conjugated to a PEG having a total molecular weight of about 40 kD.

In some cases, the reagent that may be used to generate PEGylatedaptamers is a branched PEG N-Hydroxysuccinimide (mPEG-NHS) having thegeneral formula:

with a 20 kD, 40 kD or 60 kD total molecular weight (e.g., where eachmPEG is about 10 kD, 20 kD or about 30 kD). As described above, thebranched PEGs can be linked through any appropriate reagent, such as anamino acid (e.g., lysine or glycine residues).

In one non-limiting example, the reagent used to generate PEGylatedaptamers is [N²-(monomethoxy 20 K polyethylene glycolcarbamoyl)-N⁶-(monomethoxy 20 K polyethylene glycol carbamoyl)]-lysineN-hydroxysuccinimide having the formula:

In yet another non-limiting example, the reagent used to generatePEGylated aptamers has the formula:

where X is N-hydroxysuccinimide and the PEG arms are of approximatelyequivalent molecular weight. Such PEG architecture may provide acompound with reduced viscosity compared to a similar aptamer conjugatedto a two-armed or single-arm linear PEG.

In some examples, the reagent used to generate PEGylated aptamers hasthe formula:

where X is N-hydroxysuccinimide and the PEG arms are of differentmolecular weights, for example, a 40 kD PEG of this architecture may becomposed of 2 arms of 5 kD and 4 arms of 7.5 kD. Such PEG architecturemay provide a compound with reduced viscosity compared to a similaraptamer conjugated to a two-armed PEG or a single-arm linear PEG.

In some cases, the reagent that may be used to generate PEGylatedaptamers is a non-branched mPEG-Succinimidyl Propionate (mPEG-SPA),having the general formula:

where mPEG is about 20 kD or about 30 kD. In one example, the reactiveester may be —O—CH₂—CH₂—CO₂—NHS.

In some instances, the reagent that may be used to generate PEGylatedaptamers may include a branched PEG linked through glycerol, such as theSunbright™ series from NOF Corporation, Japan. Non-limiting examples ofthese reagents include:

In another example, the reagents may include a non-branched mPEGSuccinimidyl alpha-methylbutanoate (mPEG-SMB) having the generalformula:

where mPEG is between 10 and 30 kD. In one example, the reactive estermay be —O—CH₂—CH₂—CH(CH₃)—CO₂—NHS.

In other instances, the PEG reagents may include nitrophenylcarbonate-linked PEGs, having the general formula:

Compounds including nitrophenyl carbonate can be conjugated to primaryamine containing linkers.

In some cases, the reagents used to generate PEGylated aptamers mayinclude PEG with thiol-reactive groups that can be used with athiol-modified linker. One non-limiting example may include reagentshaving the following general structure:

where mPEG is about 10 kD, about 20 kD or about 30 kD. Anothernon-limiting example may include reagents having the following generalstructure:

where each mPEG is about 10 kD, about 20 kD, or about 30 kD and thetotal molecular weight is about 20 kD, about 40 kD, or about 60 kD,respectively. Branched PEGs with thiol reactive groups that can be usedwith a thiol-modified linker, as described above, may include reagentsin which the branched PEG has a total molecular weight of about 40 kD orabout 60 kD (e.g., where each mPEG is about 20 kD or about 30 kD).

In some cases, the reagents used to generated PEGylated aptamers mayinclude reagents having the following structure:

In some cases, the reaction is carried out between about pH 6 and aboutpH 10, or between about pH 7 and pH 9 or about pH 8.

In some cases, the reagents used to generate PEGylated aptamers mayinclude reagents having the following structure:

In some cases, the reagents used to generate PEGylated aptamers mayinclude reagents having the following structure:

In some cases, the aptamer is associated with a single PEG molecule. Inother cases, the aptamer is associated with two or more PEG molecules.

In some cases, the aptamers described herein may be bound or conjugatedto one or more molecules having desired biological properties. Anynumber of molecules can be bound or conjugated to aptamers, non-limitingexamples including antibodies, peptides, proteins, carbohydrates,enzymes, polymers, drugs, small molecules, gold nanoparticles,radiolabels, fluorescent labels, dyes, haptens (e.g., biotin), otheraptamers, or nucleic acids (e.g., siRNA). In some cases, aptamers may beconjugated to molecules that increase the stability, the solubility orthe bioavailability of the aptamer. Non-limiting examples includepolyethylene glycol (PEG) polymers, carbohydrates and fatty acids. Insome cases, molecules that improve the transport or delivery of theaptamer may be used, such as cell penetration peptides. Non-limitingexamples of cell penetration peptides can include peptides derived fromTat, penetratin, polyarginine peptide Arg₈ sequence, Transportan, VP22protein from Herpes Simplex Virus (HSV), antimicrobial peptides such asBuforin I and SynB, polyproline sweet arrow peptide molecules, Pep-1 andMPG. In some embodiments, the aptamer is conjugated to a lipophiliccompound such as cholesterol, dialkyl glycerol, diacyl glycerol, or anon-immunogenic, high molecular weight compound or polymer such aspolyethylene glycol (PEG) or other water-soluble pharmaceuticallyacceptable polymers including, but not limited to, polyaminoamines(PAMAM) and polysaccharides such as dextran, or polyoxazolines (POZ).

The molecule to be conjugated can be covalently bonded or can beassociated through non-covalent interactions with the aptamer ofinterest. In one example, the molecule to be conjugated is covalentlyattached to the aptamer. The covalent attachment may occur at a varietyof positions on the aptamer, for example, to the exocyclic amino groupon the base, the 5-position of a pyrimidine nucleotide, the 8-positionof a purine nucleotide, the hydroxyl group of the phosphate, or ahydroxyl group or other group at the 5′ or 3′ terminus. In one example,the covalent attachment is to the 5′ or 3′ hydroxyl group of theaptamer.

In some cases, the aptamer can be attached to another molecule directlyor with the use of a spacer or linker. For example, a lipophiliccompound or a non-immunogenic, high molecular weight compound can beattached to the aptamer using a linker or a spacer. Various linkers andattachment chemistries are known in the art. In a non-limiting example,6-(trifluoroacetamido)hexanol(2-cyanoethyl-N,N-diisopropyl)phosphoramidite can be used to add ahexylamino linker to the 5′ end of the synthesized aptamer. This linker,as with the other amino linkers provided herein, once the groupprotecting the amine has been removed, can be reacted with PEG-NHSesters to produce covalently linked PEG-aptamers. Other non-limitingexamples of linker phosphoramidites may include: TFA-amino C4 CEDphosphoramidite having the structure:

5′-amino modifier C3 TFA having the structure:

MT amino modifier C6 CED phosphoramidite having the structure:

5′-amino modifier 5 having the structure:

5′-amino modifier C12 having the structure:

5′ thiol-modifier C6 having the structure:

5′ thiol-modifier C6 having the structure:

and 5′ thiol-modifier C6 having the structure:

The 5′-thiol modified linker may be used, for example, withPEG-maleimides, PEG-vinylsulfone, PEG-iodoacetamide andPEG-orthopyridyl-disulfide. In one example, the aptamer may be bonded tothe 5′-thiol through a maleimide or vinyl sulfone functionality.

In some cases, the aptamer formulated according to the presentdisclosure may also be modified by encapsulation within a liposome. Inother cases, the aptamer formulated according to the present disclosuremay also be modified by encapsulation within a micelle. Liposomes andmicelles may be comprised of any lipids, and in some cases the lipidsmay be phospholipids, including phosphatidylcholine.

In some cases, the aptamers described herein are designed to inhibit afunction associated with an alternative complement pathway enzyme. Inone example, an anti-fD aptamer is used to inhibit a function associatedwith fD (e.g., inhibit the catalytic activity of fD). In other cases,the aptamers described herein are designed to prevent an interaction orbinding of two or more proteins of the alternative complement pathway.In one example, an aptamer binds to fD and prevents binding of thecomplex C3bBb to fD. In another example, an aptamer of the disclosurebinds to fD and prevents binding of pre-formed C3bB complex. Theaptamers described herein may bind to a region of fD that is recognizedby an antibody or antibody fragment thereof that inhibits a functionassociated with fD. In some cases, the antibody or antibody fragmentthereof that inhibits a function associated with fD has an amino acidsequence of heavy chain variable region of:EVQLVQSGPELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGETTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCERGGVNNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHT (SEQID NO:7) and an amino acid sequence of light chain variable region of:DIQVTQSPSSLSASVGDRVTITCITSTDIDDDMNWYQQKPGKVPKLLISGGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDSLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:8).

FIG. 4 depicts modeling of the intravitreal (IVT) inhibition of Factor Dby an anti-Factor D aptamer at various IVT concentrations. FIG. 4A andFIG. 4B demonstrate IVT inhibition of Factor D at various IVTconcentrations of an anti-Factor D aptamer. Effective inhibition of IVTFactor D inhibition was modeled using a standard 2 compartment model,assuming reported IVT half-lives for Fabs (7 days, LUCENTIS®) andPEGylated aptamers (10 days, MACUGEN®) and 1:1 inhibition of Factor D byeach therapy at the relevant IVT concentrations (IC₅₀ data). As depictedin FIG. 4A, effective inhibition curves after IVT injection are shownfor an anti-Factor D Fab (dashed line), an anti-Factor D aptamer VT-001(solid line), and the intercept with the serum level of Factor D (dottedline) can be visualized as a surrogate for loss of clinically relevantFactor D inhibition. FIG. 4B depicts the predicted IVT drugconcentration (nM) of a PEGylated aptamer (dotted line) and ananti-Factor D antibody (solid line) over the number of weeks post IVTinjection.

The aptamers described herein may bind to a region of fD that isrecognized by a small molecule inhibitor that inhibits a functionassociated with fD, non-limiting examples including dichloroisocoumarinor any one of the compounds depicted in FIG. 5A, FIG. 5B, FIG. 5C, andFIG. 5D. The aptamers described herein may bind to a region of fD thatis recognized by a peptide inhibitor that inhibits a function associatedwith fD.

In some cases, an aptamer of the disclosure comprises one of thefollowing sequences described in Table 1 or Table 2.

TABLE 1 fD Aptamer Sequences SEQ ID NO. Backbone Sequence 5′ to 3′ SEQID RNA GGGAGUGUGUACGAGGCAUUAGGCCG NO: 1 CCACCCAAACUGCAGUCCUCGUAAGUCUGCCUGGCGGCUUUGAUACUUGAUCG CCCUAGAAGC SEQ ID RNAGGGAGUGUGUACGAGGCAUUAGUCCG NO: 2 CCGAAGUCUUUUGGCUCGGUUUUUUCAAGGUCGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC SEQ ID RNAGGGAGUGUGUACGAGGCAUUAGGCCG NO: 3 CCACCUCGUUUGAUUGCGGUUGUUCGGCCGCGGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC SEQ ID RNAGGGAGUGUGUACGAGGCAUUAGGCCG NO: 10 CCUUGCCAGUAUUGGCUUAGGCUGGAAGUUUGGCGGCUUUGAUACUUGAUCG CCCUAGAAGC SEQ ID RNAAGGCCGCCUUGCCAGUAUUGGCUUAG NO: 11 GCUGGAAGUUUGGCGGCUU SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 12 GGAAGUUUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 13 GGAAGUUUGGCGG SEQ ID RNACGCCUUGCAAGUAUUGGCUUUGGCUU NO: 14 GAAGUUUGGCGG SEQ ID RNACCGCCUUGCAAGUAUUGGCUUAGGCU NO: 15 UGAAGUUUGGCGG SEQ ID RNACUGCCUUGCGGGUAUUGGCGUUGGCC NO: 16 CGAAGUUUGGCUG SEQ ID RNACAGUCUUGCGAGUUUGGCUAAGCUUC NO: 17 GAAGUUUGGCUG SEQ ID RNACCGCCUUGCGAGUAAUGGCGUAGGCU NO: 18 CGAAGUUGGGCGG SEQ ID RNACCGCCUUGCGAGUAUUGGCUUAGGCU NO: 19 CGAAGUUUGGCGG SEQ ID RNACCGGCUUGCGAGUAUUGGCUUAGGCU NO: 20 CGAAGUUUGCCGG SEQ ID RNACCGCCUUGCGAGUAUUGGCUUUGGCU NO: 21 CGAAGUUUGUCUG SEQ ID RNACCGCCUUGCGAGUAUUGGCGUUGGCU NO: 22 CGAAGUUUUUCGG SEQ ID RNAUCACCUUGCCAUUAUUGGCUUAGGAU NO: 23 GGAAGUUUGGUGG SEQ ID RNACCACCUUGCCAGUAUUGGCUUUGGCC NO: 24 GGAAGUUUGGUGG SEQ ID RNACCACCUUGCCAGUAUUGGCUUAGGCU NO: 25 GGAAGUUUGGUGG SEQ ID RNACCGCCUUGCCAGUAUUGGGUUUGGCU NO: 26 GGAAGUUUGGCGG SEQ ID RNACCGCAUUGCCAGUAUUGGCUUCGGCU NO: 27 GGAAGUUUGCCGG SEQ ID RNACCGCAUUGCCAGUAUAGGCUUAUGCU NO: 28 GGAAGUUUUGCGG SEQ ID RNACCGCGUUGCCAGUAUAGGCUUAGGCU NO: 29 GGAAGUUUCGCGG SEQ ID RNACGCAUUGCUAGUAUUGGCUUAGGCUA NO: 30 GAAGUGUUGCG SEQ ID RNACUGCCUUGCCAGUAUUGGCUUAGGCU NO: 31 GGAAGUUUUUUUG SEQ ID RNACCGGGUUGCCAGUAUUGGCUUAGGCU NO: 32 GGAAGUUUCGCGG SEQ ID RNACCGACUUGCCAGUAUUGGCGUAGGCU NO: 33 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCGUAGGCU NO: 34 GGAAGUUUGCCGG SEQ ID RNACCGACUUGCCAGUAUUGGCCUAGGCU NO: 35 GGAAGUUUGUCGG SEQ ID RNAUCGACUUGCCAGUAUUGGCUUAGGCU NO: 36 GGAAGUUUGUCGG SEQ ID RNACGACUUGCCAGUAUUGGCUUAGUCUG NO: 37 GAAGUUUGUCG SEQ ID RNACCGACUUGCCAGUGUUGGCUUUGGCU NO: 38 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCUUUGGCU NO: 39 GGAAGUUUGCCGG SEQ ID RNACCGACUUGCCAGUAUUGGCUUAGGCU NO: 40 GGAAGUUUGCCGG SEQ ID RNACGGCCUUGCCAGUAUUGGCUUUGGCU NO: 41 GGAAGUUUGGCCG SEQ ID RNACGGCCUUGCAUGUAUUGGCUCAGGCA NO: 42 UGAAGUUUGUCCG SEQ ID RNACGUGCUUGUCAGUAUUGGCGUUGGCU NO: 43 GAAAGUUUGCACG SEQ ID RNACGUACUUGCCAGUAUUGGCGUAGGCU NO: 44 GGAAGUUUGUGCG SEQ ID RNACGGGCUUGCCAGUAUUAGGGUAGGCU NO: 45 GGAAGUUUGGCCG SEQ ID RNACGGGCUUGCCAGUAUUGGCUUAGGCU NO: 46 GGAAGUUUGUCCG SEQ ID RNAGAGACUCGCCAGUAUAGGCUAAGGCU NO: 47 GGAAGUAUGUCUG SEQ ID RNACUGACUUGCCAGUAUUGGCUUAGGCU NO: 48 GGAAGUUUGUCGG SEQ ID RNACUGACUUGCCAGUAUUGGCUUAGGCU NO: 49 GGAAGUUUGUCAG SEQ ID RNACUGACUUGCCAGUAUUAGCUUAGGCU NO: 50 GGAAGUUUGGCGG SEQ ID RNACCGGCUUGCCAGUAUUGGCUUAGGCU NO: 51 GGAAGUUUGCCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 52 GGAAGUUUGUCGG SEQ ID RNACCGGCUUGCCAGUAUUGGCUCAGGCU NO: 53 GGAAGUUUGCCGG SEQ ID RNACCGGCUUGCCAGUAUAGGCUCAGGCU NO: 54 GGAAGUUUGCCGG SEQ ID RNAUCGGCUUGCCAGUAUUGGCUUAGGCU NO: 55 GGAAGUUUGCCGG SEQ ID RNACCGGCUUGCCAGUAUUGGCUUUGGCU NO: 56 GGAAGUUUGCCGG SEQ ID RNACCGGCUUGCCAGUAUUGGCGUAGGCU NO: 57 GGAAGUUUGCCGG SEQ ID RNACCGACUUGCCAGUAUUCGCUUAGGCG NO: 58 GGAAGUUUGCCGG SEQ ID RNACCGACUUGCCAGUAUUGGCUUAGGCU NO: 59 GGAAGUUUGUCGG SEQ ID RNACGACUUGCCAGUAUUGGCUUAGGCUG NO: 60 GAAGUUUGUCG SEQ ID RNACCGACUUGCCAGUAAUGGCUUAGGCU NO: 61 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCGAUGGCU NO: 62 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCUUUGGCU NO: 63 GGAAGUUUGUCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 64 GGAAGUAUGGCGG SEQ ID RNACCACCUUGCCAGUAUUGGCUUAGGCU NO: 65 GGAAGUGUGGUGG SEQ ID RNAACACCUUGCGAGUAUUGGCUUAGGCU NO: 66 CGAAGUUUGGCGU SEQ ID RNAACGCUUUGCCAGUAUUGGCUUAGGCU NO: 67 GGAAGUUUCGCGU SEQ ID RNACCGCGUUGCCAGUAUUGGCUUUGGCU NO: 68 GGAAGUUUCGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 69 GGAAGUUUUGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUCAGGCU NO: 70 GGAAGUUUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUAGCGUAGGCU NO: 71 GGAAGUUUGGCGG SEQ ID RNACCGCCUUGCAAGUAUUGGCGUUGGCU NO: 72 UGAAGUUUGGCGG SEQ ID RNAUCGCCUUGCAUGUAUUGGGUUUGGCA NO: 73 UGAAGUUUGGCGG SEQ ID RNACCGCCUUGCAAGUAUAGGCUUAGGCU NO: 74 UAAGUUUGGGGG SEQ ID RNACAGCCAUGCAAGUAUUGGCUUAGGCU NO: 75 UGAAGUUCGGCUG SEQ ID RNACAGCCGUGCUAGUAUUGGCAUUGGCU NO: 76 AGAAGUUAGGCUG SEQ ID RNACGGGCUUGCCAGUAUUGGCGUUGGCU NO: 77 GGAAGUUUGCCCG SEQ ID RNAUGGGCUUGCCAGUAUUGGCUUAGGCU NO: 78 GGAAGUUUGCCCG SEQ ID RNAGGGCUUGCCAGUAUUGGCUUAGGCUG NO: 79 GAAGUUUGUCC SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 80 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGUCGCCU NO: 81 UGCAAGUAUUGGCUUUGGCUUGAAGUUUGGCGGCCUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 82 UGCAAGUAUUGGCUUAGGCUUGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCUGCCU NO: 83 UGCGGGUAUUGGCGUUGGCCCGAAGUUUGGCUGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCAGUCU NO: 84 UGCGAGUUUGGCUAAGCUUCGAAGUUUGGCUGCUUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 85 UGCGAGUAAUGGCGUAGGCUCGAAGU UGGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCGCCU NO: 86 UGCGAGUAUUGGCUUAGGCUCGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGCU NO: 87 UGCGAGUAUUGGCUUAGGCUCGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 88 UGCGAGUAUUGGCUUUGGCUCGAAGUUUGUCUGGUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 89 UGCGAGUAUUGGCGUUGGCUCGAAGU UUUUCGGCUUUGAUACUUGAUCGCCC UAGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGUCACCU NO: 90 UGCCAUUAUUGGCUUAGGAUGGAAGUUUGGUGGCUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCACCUNO: 91 UGCCAGUAUUGGCUUUGGCCGGAAGU UUGGUGGCUUUGAUACUUGAUCGCCC UAGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCACCU NO: 92 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGGUGGCUUGUGAUACUUGAUCGCC CUAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 93 UGCCAGUAUUGGGUUUGGCUGGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCAU NO: 94 UGCCAGUAUUGGCUUCGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCAU NO: 95 UGCCAGUAUAGGCUUAUGCUGGAAGUUUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCGU NO: 96 UGCCAGUAUAGGCUUAGGCUGGAAGUUUCGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGACGCAU NO: 97 UGCUAGUAUUGGCUUAGGCUAGAAGUGUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCUGCCU NO: 98 UGCCAGUAUUGGCUUAGGCUGGAAGUUUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGGU NO: 99 UGCCAGUAUUGGCUUAGGCUGGAAGUUUCGCGGUUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 100 UGCCAGUAUUGGCGUAGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 101 UGCCAGUAUUGGCGUAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 102 UGCCAGUAUUGGCCUAGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAAGUCGACU NO: 103 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGACGACU NO: 104 UGCCAGUAUUGGCUUAGUCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 105 UGCCAGUGUUGGCUUUGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 106 UGCCAGUAUUGGCUUUGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 107 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGGCCU NO: 108 UGCCAGUAUUGGCUUUGGCUGGAAGUUUGGCCGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGGCCU NO: 109 UGCAUGUAUUGGCUCAGGCAUGAAGUUUGUCCGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGUGCU NO: 110 UGUCAGUAUUGGCGUUGGCUGAAAGUUUGCACGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGUACU NO: 111 UGCCAGUAUUGGCGUAGGCUGGAAGUUUGUGCGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGGGCU NO: 112 UGCCAGUAUUAGGGUAGGCUGGAAGUUUGGCCGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGGGCU NO: 113 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGGCCGCUUUGAUACUUGAUCGCCC UAGAACA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGGAGACUNO: 114 CGCCAGUAUAGGCUAAGGCUGGAAGU AUGUCUGCUUGAUACUUGAUCGCCCU AGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGCUGACU NO: 115 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCUGACU NO: 116 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGUCAGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCUGACU NO: 117 UGCCAGUAUUAGCUUAGGCUGGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGCU NO: 118 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 119 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGCU NO: 120 UGCCAGUAUUGGCUCAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGCU NO: 121 UGCCAGUAUAGGCUCAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGUCGGCU NO: 122 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGCU NO: 123 UGCCAGUAUUGGCUUUGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGGCU NO: 124 UGCCAGUAUUGGCGUAGGCUGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 125 UGCCAGUAUUCGCUUAGGCGGGAAGUUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 126 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 127 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGUCGCUUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 128 UGCCAGUAAUGGCUUAGGCUGGAAGU UUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGCCGACU NO: 129 UGCCAGUAUUGGCGAUGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGACU NO: 130 UGCCAGUAUUGGCUUUGGCUGGAAGUUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 131 UGCCAGUAUUGGCUUAGGCUGGAAGUAUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGAGAUGGCGCUGAUCAGGCCACCUU NO: 132 GCCAGUAUUGGCUUAGGCUGGAAGUGUGGUGGCUUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGACACCUNO: 133 UGCGAGUAUUGGCUUAGGCUCGAAGU UUGGCGUCUUUGAUACUUGAUCGCCC UAGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGACGCUU NO: 134 UGCCAGUAUUGGCUUAGGCUGGAAGUUUCGCGUCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCGU NO: 135 UGCCAGUAUUGGCUUUGGCUGGAAGUUUCGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 136 UGCCAGUAUUGGCUUAGGCUGGAAGUUUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 137 UGCCAGUAUUGGCUCAGGCUGGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 138 UGCCAGUAUUAGCGUAGGCUGGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 139 UGCAAGUAUUGGCGUUGGCUUGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGUCGCCU NO: 140 UGCAUGUAUUGGGUUUGGCAUGAAGUUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCCGCCU NO: 141 UGCAAGUAUAGGCUUAGGCUUAAGUUUGGGGGCUUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA GGGAGAUGGCGCUGAUCAGGCAGCCANO: 142 UGCAAGUAUUGGCUUAGGCUUGAAGU UCGGCUGCUUUGAUACUUGAUCGCCC UAGAAGCASEQ ID RNA GGGAGAUGGCGCUGAUCAGGCAGCCG NO: 143 UGCUAGUAUUGGCAUUGGCUAGAAGUUAGGCUGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGCGGGCU NO: 144 UGCCAGUAUUGGCGUUGGCUGGAAGUUUGCCCGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGUGGGCU NO: 145 UGCCAGUAUUGGCUUAGGCUGGAAGUUUGCCCGCUUUGAUACUUGAUCGCCC UAGAAGCA SEQ ID RNAGGGAGAUGGCGCUGAUCAGGGGGCUU NO: 146 GCCAGUAUUGGCUUAGGCUGGAAGUUUGUCCGCUUUGAUACUUGAUCGCCCU AGAAGCA SEQ ID RNA CGCCUUGCCAGUAUUGGCUUAGGCUGNO: 147 GAAGUUUGGCG SEQ ID RNA GCCUUGCCAGUAUUGGCUUAGGCUGG NO: 148AAGUUUGGC SEQ ID RNA CCGCCUUGCAGUAUUGGCUUAGGCUG NO: 149 AAGUUUGGCGG SEQID RNA CCGCCUUGCCAGAUUGGCUUAGCUGG NO: 150 AAGUUUGGCGG SEQ ID RNACCGCCUUGAAGUAUUGGCUUAGGCUU NO: 151 AAGUUUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGGCUGGAAG NO: 152 UUUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCGGCUGGA NO: 153 AGUUUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUG[I-18]GGCUGG NOs: 154 AAGUUUGGCGG and 317 SEQ ID RNACCGCCUUGCCAGUAUUG[I-9]GGCUGG NOs: 155 AAGUUUGGCGG and 317 SEQ ID RNACCGCCUUGCCAGUAUUGGC[I-9]G NOs: 156 GCUGGAAGUUUGGCGG and 317 SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 157 GGAAGUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 158 GGAAGUUGGCGG SEQ ID RNACCGCCUUGCCAGUAUUGGCUUAGGCU NO: 159 GGAAGU[I-6]GGCGG SEQ ID RNACAGCCAUGCAAGUAUUGGCUUAGGCU NO: 160 UGAAGUUCGGCUG SEQ ID RNACAGCCGUGCUAGUAUUGGCAUUGGCU NO: 161 AGAAGUUAGGCUG SEQ ID RNACCGCCUUGCGAGUAAUGGCGUAGGCU NO: 162 CGAAGUUGGGCGG SEQ ID RNACCGACUUGCCAGUGUUGGCUUUGGCU NO: 163 GGAAGUUUGUCGG SEQ ID RNACAGUCUUGCGAGUUUGGCUAAGCUUC NO: 164 GAAGUUUGGCUG SEQ ID RNACUGCCUUGCGGGUAUUGGCGUUGGCC NO: 165 CGAAGUUUGGCUG SEQ ID RNACCGACUUGCCAGUAUUGGCGAUGGCU NO: 166 GGAAGUUUGUCGG SEQ ID RNACGCAUUGCUAGUAUUGGCUUAGGCUA NO: 167 GAAGUGUUGCG SEQ ID RNACGACUUGCCAGUAUUGGCGAUGGCUG NO: 237 GAAGUUUGUCG SEQ ID RNACCGACUUGCGGGUAUUGGCGAUGGCC NO: 238 CGAAGUUUGUCGG SEQ ID RNACGACUUGCGGGUAUUGGCGAUGGCCC NO: 239 GAAGUUUGUCG SEQ ID RNACUGCCUUGCCAGUAUUGGCGAUGGCU NO: 240 GGAAGUUUGGCUG SEQ ID RNACUGCCUUGCGGGUAUUGGCGAUGGCC NO: 241 CGAAGUUUGGCUG SEQ ID RNAGCCUUGCCAGUAUUGGCGAUGGCUGG NO: 242 AAGUUUGGC SEQ ID RNACCGACUUGCGGGUAUUGGCGUUGGCC NO: 243 CGAAGUUUGUCGG SEQ ID RNACGCCUUGCCAGUAUUGGCGAUGGCUG NO: 244 GAAGUUUGGCG SEQ ID RNACCGACUUGCCAGUAUAGGCUCAGGCU NO: 245 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUAGCUUAGGCU NO: 246 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCCUAGGCU NO: 247 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUAGGCUUAUGCU NO: 248 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCGCUGGCU NO: 249 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCGCAGGCU NO: 250 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCGAAGGCU NO: 251 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCUCUGGCU NO: 252 GGAAGUUUGUCGG SEQ ID RNACCGACUUGCCAGUAUUGGCUUAGGCU NO: 253 GGAAGUUUGUCGG SEQ ID RNACCGUUGUGGGUAUUGGCUUAGGCCCA NO: 254 AAGUUUCGG SEQ ID RNAGCCUUGCGGGUAUUGGCGUUGGCCCG NO: 255 AAGUUUGGC SEQ ID RNAGCCUUGCGGGUAUUGGCGAUGGCCCG NO: 256 AAGUUUGGC SEQ ID RNAGCCUUGAGGGUAUUGGCGAUGGCCCU NO: 257 AAGUUUGGC SEQ ID RNAGCCCUUGUCCGUAUUGGCUUAGGCGG NO: 258 AAAGUUUGGGC SEQ ID RNAGCGGUUGCGGGUAUUGGCGAUGGCCC NO: 259 GAAGUUUCCGC SEQ ID RNACCCUUGCCCGUAUUGGCUUAGGCGGG NO: 260 AAGUUUGGG SEQ ID RNAGGCCUUGCCCGUAUUGGCUUAGGCGG NO: 261 GAAGUUUGGCC SEQ ID RNAGGCCUUGCCCGUAUUGGCGAUGGCGG NO: 262 GAAGUUUGGCC SEQ ID RNACGACUUGCCAGUAUUGGCGAUGGCUG NO: 263 GAAGUUUGUCG SEQ ID RNACGCCUUGCCAGUAUUGGCUUAGGCUG NO: 264 GAAGUUUGGCG SEQ ID RNACGACUUGCCAGUAUUGGCUUAGGCUG NO: 265 GAAGUUUGUCG SEQ ID RNACCGCCUUGCCAGUAUUGGCGAUGGCU NO: 266 GGAAGUUUGGCGG

TABLE 2 fD Aptamer Sequences Compound SEQ ID NO. Name Backbone Sequence5′ to 3′ SEQ ID C1 RNA GGGAGUGUGUACGAGGCAUUAGGCCG NO: 1CCACCCAAACUGCAGUCCUCGUAAGUC with UGCCUGGCGGCUUUGAUACUUGAUCGmodifications CCCUAGAAGC; where G is 2′F and A, C and U are 2′OMemodified RNA SEQ ID C2 RNA GGGAGUGUGUACGAGGCAUUAGUCCG NO: 2 withCCGAAGUCUUUUGGCUCGGUUUUUUC modifications AAGGUCGGCGGCUUUGAUACUUGAUCGCCCUAGAAGC; where G is 2′F and A, C and U are 2′OMe modified RNA SEQ IDC3 RNA GGGAGUGUGUACGAGGCAUUAGGCCG NO: 3 with CCACCUCGUUUGAUUGCGGUUGUUCGmodifications GCCGCGGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC; where G is 2′F andA, C and U are 2′OMe modified RNA SEQ ID 10FR14 RNAGGGAGUGUGUACGAGGCAUUAGGCCG NO: 10 with CCUUGCCAGUAUUGGCUUAGGCUGGAmodifications AGUUUGGCGGCUUUGAUACUUGAUCG CCCUAGAAGC; where G is 2′F andA, C and U are 2′OMe modified RNA SEQ ID S31 RNA C6S- NO: 267 withAGGCCGCCUUGCCAGUAUUGGCUUAG modifications GCUGGAAGUUUGGCGGCUUidT; where Gis 2′F and A, C and U are 2′OMe modified RNA, C6S represents asix-carbon disulfide linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID S32 RNA C6S- NO: 268 withCCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGGCGGidT; where G is2′F and A, C and U are 2′OMe modified RNA, C6S represents a six-carbondisulfide linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 15 RNA C6NH₂- NO: 269 withCCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGGCGGidT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a 6-carbonamino containing linker, and idT represents a 3′inverted deoxythymidineresidue. SEQ ID Rd3-06 RNA CGCCUUGCAAGUAUUGGCUUUGGCUU NO: 14 withGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-28 RNA CCGCCUUGCAAGUAUUGGCUUAGGCU NO: 15 withUGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-11 RNA CUGCCUUGCGGGUAUUGGCGUUGGCC NO: 16 withCGAAGUUUGGCUG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-10 RNA CAGUCUUGCGAGUU.UGGCUAAGCUUC NO: 17 withGAAGUUUGGCUG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-42 RNA CCGCCUUGCGAGUAAUGGCGUAGGCU NO: 18 withCGAAGUUGGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-18 RNA CCGCCUUGCGAGUAUUGGCUUAGGCU NO: 19 withCGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-02 RNA CCGGCUUGCGAGUAUUGGCUUAGGCU NO: 20 withCGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-46 RNA CCGCCUUGCGAGUAUUGGCUUUGGCU NO: 21 withCGAAGUUUGUCUG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-21 RNA CCGCCUUGCGAGUAUUGGCGUUGGCU NO: 22 withCGAAGUUUUUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-08 RNA UCACCUUGCCAUUAUUGGCUUAGGAU NO: 23 withGGAAGUUUGGUGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-22 RNA CCACCUUGCCAGUAUUGGCUUUGGCC NO: 24 withGGAAGUUUGGUGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-29 RNA CCACCUUGCCAGUAUUGGCUUAGGCU NO: 25 withGGAAGUUUGGUGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-35 RNA CCGCCUUGCCAGUAUUGGGUUUGGCU NO: 26 withGGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-24 RNA CCGCAUUGCCAGUAUUGGCUUCGGCU NO: 27 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-01 RNA CCGCAUUGCCAGUAUAGGCUUAUGCU NO: 28GGAAGUUUUGCGG; with where G is 2′F and A, C and U are 2′OMemodifications modified RNA. SEQ ID Rd4-07 RNA CCGCGUUGCCAGUAUAGGCUUAGGCUNO: 29 with GGAAGUUUCGCGG; modifications where G is 2′F and A, C and Uare 2′OMe modified RNA. SEQ ID Rd3-47 RNA CGCAUUGCUAGUAUUGGCUUAGGCUA NO:30 with GAAGUGUUGCG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-31 RNA CUGCCUUGCCAGUAUUGGCUUAGGCU NO: 31with GGAAGUUUUGCGG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-18 RNA CCGGGUUGCCAGUAUUGGCUUAGGCU NO: 32with GGAAGUUUCGCGG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-03 RNA CCGACUUGCCAGUAUUGGCGUAGGCU NO: 33with GGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-23 RNA CCGACUUGCCAGUAUUGGCGUAGGCU NO: 34with GGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-24 RNA CCGACUUGCCAGUAUUGGCCUAGGCU NO: 35with GGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-32 RNA UCGACUUGCCAGUAUUGGCUUAGGCU NO: 36with GGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-39 RNA CGACUUGCCAGUAUUGGCUUAGUCUG NO: 37with GAAGUUUGUCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-20 RNA CCGACUUGCCAGUGUUGGCUUUGGCU NO: 38 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-38 RNA CCGACUUGCCAGUAUUGGCUUUGGCU NO: 39 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-13 RNA CCGACUUGCCAGUAUUGGCUUAGGCU NO: 40 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-37 RNA CGGCCUUGCCAGUAUUGGCUUUGGCU NO: 41 withGGAAGUUUGGCCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-03 RNA CGGCCUUGCAUGUAUUGGCUCAGGCA NO: 42 withUGAAGUUUGUCCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-25 RNA CGUGCUUGUCAGUAUUGGCGUUGGCU NO: 43 withGAAAGUUUGCACG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-27 RNA CGUACUUGCCAGUAUUGGCGUAGGCU NO: 44 withGGAAGUUUGUGCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-47 RNA CGGGCUUGCCAGUAUUAGGGUAGGCU NO: 45 withGGAAGUUUGGCCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-12 RNA CGGGCUUGCCAGUAUUGGCUUAGGCU NO: 46 withGGAAGUUUGUCCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-33 RNA GAGACUCGCCAGUAUAGGCUAAGGCU NO: 47 withGGAAGUAUGUCUG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-39 RNA CUGACUUGCCAGUAUUGGCUUAGGCU NO: 48 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-10 RNA CUGACUUGCCAGUAUUGGCUUAGGCU NO: 49 withGGAAGUUUGUCAG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-25 RNA CUGACUUGCCAGUAUUAGCUUAGGCU NO: 50 withGGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-16 RNA CCGGCUUGCCAGUAUUGGCUUAGGCU NO: 51 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-21 RNA CCGCCUUGCCAGUAUUGGCUUAGGCU NO: 52 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-20 RNA CCGGCUUGCCAGUAUUGGCUCAGGCU NO: 53 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-41 RNA CCGGCUUGCCAGUAUAGGCUCAGGCU NO: 54 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-40 RNA UCGGCUUGCCAGUAUUGGCUUAGGCU NO: 55 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-02 RNA CCGGCUUGCCAGUAUUGGCUUUGGCU NO: 56 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-43 RNA CCGGCUUGCCAGUAUUGGCGUAGGCU NO: 57 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-34 RNA CCGACUUGCCAGUAUUCGCUUAGGCG NO: 58 withGGAAGUUUGCCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-04 RNA CCGACUUGCCAGUAUUGGCUUAGGCU NO: 59 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-36 RNA CGACUUGCCAGUAUUGGCUUAGGCUG NO: 60 withGAAGUUUGUCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-06 RNA CCGACUUGCCAGUAAUGGCUUAGGCU NO: 61 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-13 RNA CCGACUUGCCAGUAUUGGCGAUGGCU NO: 62 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-15 RNA CCGACUUGCCAGUAUUGGCUUUGGCU NO: 63 withGGAAGUUUGUCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-16 RNA CCGCCUUGCCAGUAUUGGCUUAGGCU NO: 64 withGGAAGUAUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-01 RNA CCACCUUGCCAGUAUUGGCUUAGGCU NO: 65 withGGAAGUGUGGUGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-26 RNA ACACCUUGCGAGUAUUGGCUUAGGCU NO: 66 withCGAAGUUUGGCGU; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-07 RNA ACGCUUUGCCAGUAUUGGCUUAGGCU NO: 67 withGGAAGUUUCGCGU; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-23 RNA CCGCGUUGCCAGUAUUGGCUUUGGCU NO: 68 withGGAAGUUUCGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-26 RNA CCGCCUUGCCAGUAUUGGCUUAGGCU NO: 69 withGGAAGUUUUGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-30 RNA CCGCCUUGCCAGUAUUGGCUCAGGCU NO: 70 withGGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-41 RNA CCGCCUUGCCAGUAUUAGCGUAGGCU NO: 71 withGGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-11 RNA CCGCCUUGCAAGUAUUGGCGUUGGCU NO: 72 withUGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-43 RNA UCGCCUUGCAUGUAUUGGGUUUGGCA NO: 73 withUGAAGUUUGGCGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-28 RNA CCGCCUUGCAAGUAUAGGCUUAGGCU NO: 74 withUAAGUUUGGGGG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-34 RNA CAGCCAUGCAAGUAUUGGCUUAGGCU NO: 75 withUGAAGUUCGGCUG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd4-19 RNA CAGCCGUGCUAGUAUUGGCAUUGGCU NO: 76 withAGAAGUUAGGCUG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-44 RNA CGGGCUUGCCAGUAUUGGCGUUGGCU NO: 77 withGGAAGUUUGCCCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-14 RNA UGGGCUUGCCAGUAUUGGCUUAGGCU NO: 78 withGGAAGUUUGCCCG; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID Rd3-15 RNA GGGCUUGCCAGUAUUGGCUUAGGCUG NO: 79 withGAAGUUUGUCC; modifications where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID 10F/R-14 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCU NO: 80with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-06 full RNA GGGAGAUGGCGCUGAUCAGGUCGCCUNO: 81 with length UGCAAGUAUUGGCUUUGGCUUGAAGU modificationsUUGGCGGCCUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-28 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 82 with length UGCAAGUAUUGGCUUAGGCUUGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-11 full RNA GGGAGAUGGCGCUGAUCAGGCUGCCUNO: 83 with length UGCGGGUAUUGGCGUUGGCCCGAAGU modificationsUUGGCUGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-10 full RNA GGGAGAUGGCGCUGAUCAGGCAGUCUNO: 84 with length UGCGAGUUUGGCUAAGCUUCGAAGUU modificationsUGGCUGCUUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-42 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 85 with length UGCGAGUAAUGGCGUAGGCUCGAAGU modificationsUGGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-18 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 86 with length UGCGAGUAUUGGCUUAGGCUCGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-02 full RNA GGGAGAUGGCGCUGAUCAGGCCGGCUNO: 87 with length UGCGAGUAUUGGCUUAGGCUCGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-46 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 88 with length UGCGAGUAUUGGCUUUGGCUCGAAGU modificationsUUGUCUGGUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-21 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 89 with length UGCGAGUAUUGGCGUUGGCUCGAAGU modificationsUUUUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-08 full RNA GGGAGAUGGCGCUGAUCAGGUCACCUNO: 90 with length UGCCAUUAUUGGCUUAGGAUGGAAGU modificationsUUGGUGGCUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-22 full RNA GGGAGAUGGCGCUGAUCAGGCCACCUNO: 91 with length UGCCAGUAUUGGCUUUGGCCGGAAGU modificationsUUGGUGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-29 full RNA GGGAGAUGGCGCUGAUCAGGCCACCUNO: 92 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGGUGGCUUGUGAUACUUGAUCGCC CUAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-35 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 93 with length UGCCAGUAUUGGGUUUGGCUGGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-24 full RNA GGGAGAUGGCGCUGAUCAGGCCGCAUNO: 94 with length UGCCAGUAUUGGCUUCGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-01 full RNA GGGAGAUGGCGCUGAUCAGGCCGCAUNO: 95 with length UGCCAGUAUAGGCUUAUGCUGGAAGU modificationsUUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-07 full RNA GGGAGAUGGCGCUGAUCAGGCCGCGUNO: 96 with length UGCCAGUAUAGGCUUAGGCUGGAAGU modificationsUUCGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-47 full RNA GGGAGAUGGCGCUGAUCAGGACGCAUNO: 97 with length UGCUAGUAUUGGCUUAGGCUAGAAGU modificationsGUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-31 full RNA GGGAGAUGGCGCUGAUCAGGCUGCCUNO: 98 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-18 full RNA GGGAGAUGGCGCUGAUCAGGCCGGGUNO: 99 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUCGCGGUUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-03 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 100 with length UGCCAGUAUUGGCGUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-23 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 101 with length UGCCAGUAUUGGCGUAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-24 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 102 with length UGCCAGUAUUGGCCUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-32 full RNA GGGAGAUGGCGCUGAUCAAGUCGACUNO: 103 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-39 full RNA GGGAGAUGGCGCUGAUCAGGACGACUNO: 104 with length UGCCAGUAUUGGCUUAGUCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-20 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 105 with length UGCCAGUGUUGGCUUUGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-38 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 106 with length UGCCAGUAUUGGCUUUGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-13 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 107 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-37 full RNA GGGAGAUGGCGCUGAUCAGGCGGCCUNO: 108 with length UGCCAGUAUUGGCUUUGGCUGGAAGU modificationsUUGGCCGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-03 full RNA GGGAGAUGGCGCUGAUCAGGCGGCCUNO: 109 with length UGCAUGUAUUGGCUCAGGCAUGAAGU modificationsUUGUCCGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-25 full RNA GGGAGAUGGCGCUGAUCAGGCGUGCUNO: 110 with length UGUCAGUAUUGGCGUUGGCUGAAAGU modificationsUUGCACGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-27 full RNA GGGAGAUGGCGCUGAUCAGGCGUACUNO: 111 with length UGCCAGUAUUGGCGUAGGCUGGAAGU modificationsUUGUGCGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-47 full RNA GGGAGAUGGCGCUGAUCAGGCGGGCUNO: 112 with length UGCCAGUAUUAGGGUAGGCUGGAAGU modificationsUUGGCCGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-12 full RNA GGGAGAUGGCGCUGAUCAGGCGGGCUNO: 113 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGGCCGCUUUGAUACUUGAUCGCCC UAGAACA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-33 full RNA GGGAGAUGGCGCUGAUCAGGGAGACUNO: 114 with length CGCCAGUAUAGGCUAAGGCUGGAAGU modificationsAUGUCUGCUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-39 full RNA GGGAGAUGGCGCUGAUCAGGCUGACUNO: 115 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-10 full RNA GGGAGAUGGCGCUGAUCAGGCUGACUNO: 116 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGUCAGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-25 full RNA GGGAGAUGGCGCUGAUCAGGCUGACUNO: 117 with length UGCCAGUAUUAGCUUAGGCUGGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-16 full RNA GGGAGAUGGCGCUGAUCAGGCCGGCUNO: 118 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-21 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 119 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-20 full RNA GGGAGAUGGCGCUGAUCAGGCCGGCUNO: 120 with length UGCCAGUAUUGGCUCAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-41 full RNA GGGAGAUGGCGCUGAUCAGGCCGGCUNO: 121 with length UGCCAGUAUAGGCUCAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-40 full RNA GGGAGAUGGCGCUGAUCAGGUCGGCUNO: 122 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-02 full RNA GGGAGAUGGCGCUGAUCAGGCCGGCUNO: 123 with length UGCCAGUAUUGGCUUUGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-43 full RNA GGGAGAUGGCGCUGAUCAGGCCGGCUNO: 124 with length UGCCAGUAUUGGCGUAGGCUGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-34 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 125 with length UGCCAGUAUUCGCUUAGGCGGGAAGU modificationsUUGCCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-04 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 126 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-36 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 127 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGUCGCUUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-06 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 128 with length UGCCAGUAAUGGCUUAGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-13 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 129 with length UGCCAGUAUUGGCGAUGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-15 full RNA GGGAGAUGGCGCUGAUCAGGCCGACUNO: 130 with length UGCCAGUAUUGGCUUUGGCUGGAAGU modificationsUUGUCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-16 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 131 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsAUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-01 full RNA GGAGAUGGCGCUGAUCAGGCCACCUUNO: 132 with length GCCAGUAUUGGCUUAGGCUGGAAGUG modificationsUGGUGGCUUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-26 full RNA GGGAGAUGGCGCUGAUCAGGACACCUNO: 133 with length UGCGAGUAUUGGCUUAGGCUCGAAGU modificationsUUGGCGUCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-07 full RNA GGGAGAUGGCGCUGAUCAGGACGCUUNO: 134 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUCGCGUCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-23 full RNA GGGAGAUGGCGCUGAUCAGGCCGCGUNO: 135 with length UGCCAGUAUUGGCUUUGGCUGGAAGU modificationsUUCGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-26 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 136 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUUGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-30 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 137 with length UGCCAGUAUUGGCUCAGGCUGGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-41 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 138 with length UGCCAGUAUUAGCGUAGGCUGGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-11 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 139 with length UGCAAGUAUUGGCGUUGGCUUGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-43 full RNA GGGAGAUGGCGCUGAUCAGGUCGCCUNO: 140 with length UGCAUGUAUUGGGUUUGGCAUGAAGU modificationsUUGGCGGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-28 full RNA GGGAGAUGGCGCUGAUCAGGCCGCCUNO: 141 with length UGCAAGUAUAGGCUUAGGCUUAAGUU modificationsUGGGGGCUUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-34 full RNA GGGAGAUGGCGCUGAUCAGGCAGCCANO: 142 with length UGCAAGUAUUGGCUUAGGCUUGAAGU modificationsUCGGCUGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd4-19 full RNA GGGAGAUGGCGCUGAUCAGGCAGCCGNO: 143 with length UGCUAGUAUUGGCAUUGGCUAGAAGU modificationsUAGGCUGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-44 full RNA GGGAGAUGGCGCUGAUCAGGCGGGCUNO: 144 with length UGCCAGUAUUGGCGUUGGCUGGAAGU modificationsUUGCCCGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-14 full RNA GGGAGAUGGCGCUGAUCAGGUGGGCUNO: 145 with length UGCCAGUAUUGGCUUAGGCUGGAAGU modificationsUUGCCCGCUUUGAUACUUGAUCGCCC UAGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Rd3-15 full RNA GGGAGAUGGCGCUGAUCAGGGGGCUUNO: 146 with length GCCAGUAUUGGCUUAGGCUGGAAGUU modificationsUGUCCGCUUUGAUACUUGAUCGCCCU AGAAGCA; where G is 2′F and A, C and U are2′OMe modified RNA. SEQ ID Aptamer 16 RNA C6NH₂- NO: 270 withCGCCUUGCCAGUAUUGGCUUAGGCUG modifications GAAGUUUGGCG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 17 RNA C6NH₂- NO: 271 withGCCUUGCCAGUAUUGGCUUAGGCUGG modifications AAGUUUGGC-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 18 RNA C6NH₂- NO: 272 withCCGCCUUGCAGUAUUGGCUUAGGCUG modifications AAGUUUGGCGG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 19 RNA C6NH₂- NO: 273 withCCGCCUUGCCAGAUUGGCUUAGCUGG modifications AAGUUUGGCGG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 20 RNA C6NH₂- NO: 274 withCCGCCUUGAAGUAUUGGCUUAGGCUU modifications AAGUUUGGCGG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 21 RNA C6NH₂- NO: 275 withCCGCCUUGCCAGUAUUGGGCUGGAAG modifications UUUGGCGG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 22 RNA C6NH₂- NO: 276 withCCGCCUUGCCAGUAUUGGCGGCUGGA modifications AGUUUGGCGG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 23 RNA C6NH₂-CCGCCUUGCCAGUAUUG[I- NOs: 15418]GGCUGGAAGUUUGGCGG-idT; and 318 with where G is 2′F and A, C and U are2′OMe modifications modified RNA, [I-18] represents a hexaethyleneglycol spacer, C6NH₂ represents a six-carbon amino containing linker,and idT represents a 3′ inverted deoxythymidine residue. SEQ ID Aptamer24 RNA C6NH₂-CCGCCUUGCCAGUAUUG[I- NOs: 155 9]GGCUGGAAGUUUGGCGG-idT; and318 with where G is 2′F and A, C and U are 2′OMe modifications modifiedRNA, [I-9] represents a 9-carbon spacer, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 25 RNA C6NH₂-CCGCCUUGCCAGUAUUGGC[I- NOs: 1569]GGCUGGAAGUUUGGCGG-idT; and 318 with where G is 2′F and A, C and U are2′OMe modifications modified RNA, [I-9] represents a 9-carbon spacer,C6NH₂ represents a six-carbon amino containing linker, and idTrepresents a 3′ inverted deoxythymidine residue. SEQ ID Aptamer 26 RNAC6NH₂- NO: 277 with CCGCCUUGCCAGUAUUGGCUUAGGCU modificationsGGAAGUGGCGG-idT; where G is 2′F and A, C and U are 2′OMe modified RNA,C6NH₂ represents a six-carbon amino containing linker, and idTrepresents a 3′ inverted deoxythymidine residue. SEQ ID Aptamer 27 RNAC6NH₂- NO: 278 with CCGCCUUGCCAGUAUUGGCUUAGGCU modificationsGGAAGUUGGCGG-idT; where G is 2′F and A, C and U are 2′OMe modified RNA,C6NH₂ represents a six-carbon amino containing linker, and idTrepresents a 3′ inverted deoxythymidine residue. SEQ ID Aptamer 28 RNAC6NH₂- NO: 159 with CCGCCUUGCCAGUAUUGGCUUAGGCU modificationsGGAAGU[I-6]GGCGG-idT; where G is 2′F and A, C and U are 2′OMe modifiedRNA, [I-6] represents a 6-carbon spacer, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 33 RNA C6NH₂- NO: 279 withCAGCCAUGCAAGUAUUGGCUUAGGCU modifications UGAAGUUCGGCUG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 34 RNA C6NH₂- NO: 280 withCAGCCGUGCUAGUAUUGGCAUUGGCU modifications AGAAGUUAGGCUG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 35 RNA C6NH₂- NO: 281 withCCGCCUUGCGAGUAAUGGCGUAGGCU modifications CGAAGUUGGGCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 36 RNA C6NH₂- NO: 282 withCCGACUUGCCAGUGUUGGCUUUGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 37 RNA C6NH₂- NO: 283 withCAGUCUUGCGAGUUUGGCUAAGCUUC modifications GAAGUUUGGCUG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 38 RNA C6NH₂- NO: 284 withCUGCCUUGCGGGUAUUGGCGUUGGCC modifications CGAAGUUUGGCUG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 39 RNA C6NH₂- NO: 285 withCCGACUUGCCAGUAUUGGCGAUGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 40 RNA C6NH₂- NO: 286 withCGCAUUGCUAGUAUUGGCUUAGGCUA modifications GAAGUGUUGCG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 53 RNA C6NH₂- NO: 287 withCGACUUGCCAGUAUUGGCGAUGGCUG modifications GAAGUUUGUCG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 54 RNA C6NH₂- NO: 288 with CCGACUUGCGGGUAUUGGCGAUGGCCmodifications CGAAGUUUGUCGG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 55 RNA C6NH₂- NO: 289with CGACUUGCGGGUAUUGGCGAUGGCCC modifications GAAGUUUGUCG-idT; where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 56 RNA C6NH₂- NO: 290 withCUGCCUUGCCAGUAUUGGCGAUGGCU modifications GGAAGUITUGGCUG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 57 RNA C6NH₂- NO: 291 with CUGCCUUGCGGGUAUUGGCGAUGGCCmodifications CGAAGUUUGGCUG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 58 RNA C6NH₂- NO: 292with GCCUUGCCAGUAUUGGCGAUGGCUGG modifications AAGUUUGGC-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 59 RNA C6NH₂- NO: 293 with CCGACUUGCGGGUAUUGGCGUUGGCCmodifications CGAAGUUUGUCGG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 60 RNA C6NH₂- NO: 294with CGCCUUGCCAGUAUUGGCGAUGGCUG modifications GAAGUUUGGCG-idT; where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 62 RNA C6NH₂- NO: 295 withCCGACUUGCCAGUAUAGGCUCAGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 64 RNA C6NH₂- NO: 296 with CCGACUUGCCAGUAUUAGCUUAGGCUmodifications GGAAGUUUGUCGG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 66 RNA C6NH₂- NO: 297with CCGACUUGCCAGUAUUGGCCUAGGCU modifications GGAAGUUUGUCGG-idT; where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 67 RNA C6NH₂- NO: 298 withCCGACUUGCCAGUAUAGGCUUAUGCU modifications GGAAGUUUGUCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 68 RNA C6NH₂- NO: 299 with CCGACUUGCCAGUAUUGGCGCUGGCUmodifications GGAAGUUUGUCGG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 69 RNA C6NH₂- NO: 300with CCGACUUGCCAGUAUUGGCGCAGGCU modifications GGAAGUUUGUCGG-idT; where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 71 RNA C6NH₂- NO: 301 withCCGACUUGCCAGUAUUGGCGAAGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 72 RNA C6NH₂- NO: 302 with CCGACUUGCCAGUAUUGGCUCUGGCUmodifications GGAAGUUUGUCGG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 74 RNA C6NH₂- NO: 269with CCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGGCGG-idT; where Gis 2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 76 RNA C6NH₂- NO: 285 withCCGACUUGCCAGUAUUGGCGAUGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 88 RNA C6NH₂- NO: 303 withCCGACUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 90 RNA C6NH₂- NO: 304 with CCGUUGUGGGUAUUGGCUUAGGCCCAmodifications AAGUUUCGG-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 91 RNA C6NH₂- NO: 305with GCCUUGCGGGUAUUGGCGUUGGCCCG modifications AAGUUUGGC-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 92 RNA C6NH₂- NO: 306 with GCCUUGCGGGUAUUGGCGAUGGCCCGmodifications AAGUUUGGC-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 93 RNA C6NH₂- NO: 307with GCCUUGAGGGUAUUGGCGAUGGCCCU modifications AAGUUUGGC-idT; where G is2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 94 RNA C6NH₂- NO: 308 with GCCCUUGUCCGUAUUGGCUUAGGCGGmodifications AAAGUUUGGGC-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 95 RNA C6NH₂- NO: 309with GCGGUUGCGGGUAUUGGCGAUGGCCC modifications GAAGUUUCCGC-idT; where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 96 RNA C6NH₂- NO: 310 withCCCUUGCCCGUAUUGGCUUAGGCGGG modifications AAGUUUGGG-idT; where G is 2′Fand A, C and U are 2′OMe modified RNA; C6NH₂ represents a hexylaminelinker; and idT represents a 3′ inverted deoxythymidine residue. SEQ IDAptamer 97 RNA C6NH₂- NO: 311 with GGCCUUGCCCGUAUUGGCUUAGGCGGmodifications GAAGUUUGGCC-idT; where G is 2′F and A, C and U are 2′OMemodified RNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. SEQ ID Aptamer 98 RNA C6NH₂- NO: 312with GGCCUUGCCCGUAUUGGCGAUGGCGG modifications GAAGUUUGGCC-idT; where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 99 RNA C6NH₂- NO: 294 withCGCCUUGCCAGUAUUGGCGAUGGCUG modifications GAAGUUUGGCG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 102 RNA C6NH₂- NO: 313 withCGACUUGCCAGUAUUGGCGAUGGCUG modifications GAAGUUUGUCG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 104 RNA C6NH₂- NO: 314 withCGCCUUGCCAGUAUUGGCUUAGGCUG modifications GAAGUUUGGCG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 106 RNA C6NH₂- NO: 303 withCCGACUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGUCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 107 RNA C6NH₂- NO: 315 withCGACUUGCCAGUAUUGGCUUAGGCUG modifications GAAGUUUGUCG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 108 RNA C6NH₂- NO: 306 withGCCUUGCGGGUAUUGGCGAUGGCCCG modifications AAGUUUGGC-idT; where G is 2′F,and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents a six-carbonamino containing linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer 109 RNA C6NH₂- NO: 312 withGGCCUUGCCCGUAUUGGCGAUGGCGG modifications GAAGUUUGGCC-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer 116 RNA C6NH₂- NO: 316 withCCGCCUUGCCAGUAUUGGCGAUGGCU modifications GGAAGUUUGGCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, and idT represents a 3′ inverteddeoxythymidine residue. SEQ ID Aptamer RNA C6SH- NO: 269 with 1721CCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUHUGGCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6SH represents asix-carbon thiol linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer RNA C6SH- NO: 269 with 1722CCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGGCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6SH represents asix-carbon thiol linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer RNA C6SH- NO: 269 with 1723CCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGGCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6SH represents asix-carbon thiol linker, and idT represents a 3′ inverted deoxythymidineresidue. SEQ ID Aptamer RNA C6SH- NO: 269 with 1724CCGCCUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGGCGG-idT; where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6SH represents asix-carbon thiol linker, and idT represents a 3′ inverted deoxythymidineresidue.

In some cases, an aptamer of the disclosure has a nucleic acid sequencethat comprises SEQ ID NOs:13 or 269. In some cases, an aptamer having anucleic acid sequence comprising SEQ ID NOs:13 or 269 inhibits afunction associated with fD. In some cases, an aptamer having a nucleicacid sequence comprising SEQ ID NOs:13 or 269 binds to and/or blocksaccess to the active site of fD. In some cases, an aptamer having anucleic acid sequence comprising SEQ ID NOs:13 or 269 binds to and/orblocks access to the exosite of fD. In some cases, an aptamer having anucleic acid sequence comprising SEQ ID NOs:13 or 269 prevents orreduces binding of pre-formed C3bB complex to fD.

In some aspects, an aptamer of the disclosure has a nucleic acidsequence comprising any one of SEQ ID NOs:13, 165, 166, 244, 253, 256,262, 269, 284, 285, 294, 303, 306, and 312, or having at least 80%sequence identity to any one of SEQ ID NOs:13, 165, 166, 244, 253, 256,262, 269, 284, 285, 294, 303, 306, and 312. In some cases, the nucleicacid sequence comprises one or more modified nucleotides. In some cases,at least 50% of said nucleic acid sequence comprises the one or moremodified nucleotides. In some cases, the one or more modifiednucleotides comprises a 2′F-modified nucleotide, a 2′OMe-modifiednucleotide, or a combination thereof. In some cases, the one or moremodified nucleotides are selected from the group consisting of: 2′F-G,2′OMe-G, 2′OMe-U, 2′OMe-A, 2′OMe-C, an inverted deoxythymidine at the 3′terminus, and any combination thereof. In some cases, aptamer isselected from the group consisting of: Aptamer 76 as described in Table2, Aptamer 116 as described in Table 2, Aptamer 102 as described inTable 2, Aptamer 104 as described in Table 2, Aptamer 106 as describedin Table 2, Aptamer 108 as described in Table 2, Aptamer 107 asdescribed in Table 2, Aptamer 109 as described in Table 2, and Aptamer99 as described in Table 2. In some cases, the aptamer is conjugated toa polyethylene glycol (PEG) molecule. In some cases, the PEG moleculehas a molecular weight of 80 kDa or less (e.g., 40 kDa). In some cases,the PEG molecule is conjugated to the aptamer using a pegylationreagent. In some cases, the pegylation reagent comprises2,3-Bis(methylpolyoxyethylene-oxy)-1-{3-[(1,5-dioxo-5-succinimidyloxy,pentyl)amino]propyloxy} propane.

In some cases, an aptamer of the disclosure does not comprise any one ofSEQ ID NOs:1-3, 168-235 as described in Table 3. In some cases, anaptamer of the disclosure does not comprise any one of SEQ IDNOs:168-235 as described in Table 3. In some cases, an aptamer of thedisclosure does not comprise any one of SEQ ID NOs:228-235 as describedin Table 3.

TABLE 3 fD Aptamer Sequences SEQ ID NO. Backbone Sequence 5′ to 3′ SEQID RNA GGGAGUGUGUACGAGGCAUUAGGCCG NO: 1 CCACCCAAACUGCAGUCCUCGUAAGUCUGCCUGGCGGCUUUGAUACUUGAUCG CCCUAGAAGC; where G is 2′F and A, C and U are2′OMe modified RNA SEQ ID RNA GGGAGUGUGUACGAGGCAUUAGUCCG NO: 2CCGAAGUCUUUUGGCUCGGUUUUUUC AAGGUCGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC; whereG is 2′F and A, C and U are 2′OMe modified RNA SEQ ID RNAGGGAGUGUGUACGAGGCAUUAGGCCG NO: 3 CCACCUCGUUUGAUUGCGGUUGUUCGGCCGCGGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC; where G is 2′F and A, C and Uare 2′OMe modified RNA SEQ ID DNA GTGACGACTGACATATCTGCTCCGAGG NO: 168TTATTGGGGTTGGGGCCTGGGCGATTG GGGCCTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGTTTGGG NO: 169 GTTGGGGCCTGGGAGTTTGGGGAGCAGAAAGGACGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT NO:170 GTTGTGGGGGTGGGTGGTGGGCCCTTC GCCATGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGGCGGTT NO: 171 GGGGTCGAAGGGCGAGGGGTGGGAGGTCGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTATTTTGG NO: 172GGCCTGGGTGTTGGGGATTGGGGACTA TGTGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGTGGAT NO: 173 GGTGGGGGGTGGTGTGGGAGGGCTGGTCGGTCGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCCCTATAG NO:174 GGGTGTGGGCGAGGGGTGGGTGGTAGG GCGGCTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGGAGGTG NO: 175 GGTGGGTGGGTGCGTGCGAGGGCGGTGTAGGTCCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCAAAAGTT NO:176 AGATTGACATGGTATGCACCGTCTGAG GTTGGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCACCACGC NO: 177 TAGGGGTGAGGGCGAGGGGTGGGTAGCGCGTGGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT NO:178 GTTGTGGGGGCGGGTGGTGGGTGCGTC GGTGGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGCTTCCA NO: 179 GCGGTCATGATATGCACTGTCTGAAGCTCGGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTCTGTTAT NO:180 GATATGCACCGTCTGAGGGTAGTCGCG GGGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGCTTGTT NO: 181 TAGTGGGTGGGTGGGTGGTGTGGTGGTGATGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCCTTGGGG NO: 182TTGGGGCCTGGGTGTTTGGGGTGGCCT AGAAGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGCTAGGG NO: 183 GTGGGTTGGGGTTGGTGGTGTGCGTGTGGGTTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTTGAG NO:184 GTTGGTGGGGGGTGGGCGGTGGGATGG TTGTGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTTGACAG NO: 185 TCTGCTTTGCAGGGGCCGAGAGCGCCATTGCGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTGGTTG NO:186 GTGGGGGGTGGAGGGTGGGAGGCCGTG TGTCCCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGTGGTG NO: 187 GTGGGGGAGGGTGGTGGGGTGGCCGGCGCTCGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGGGTTA NO:188 CGTGGTTCGGGGCTAGGGGGGTGGGGT GTGTTTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGGTGGT NO: 189 GTGCGGTGGGTTCTTGGGTGGGATGGGTGGTACCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTATTAGAT NO:190 CCTCGGTGGGTGGGTGGGTGTGTGGTG GTGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGGGCGTC NO: 191 TGAGCGCATGGATGACCCACCGACAGATTGCGGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCGCTTTGG NO:192 GTGGGCTCGGTGTGCGGTGTGCGGGTG GGTTTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGTTTGGG NO: 193 GTTGGGGCCTGGGAGTTTGGGGAGCAGAAAGGGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCGGGTGGG NO:194 TTGGGTTGGGTTTGGTGGTGGTGCCTGT TAGTTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCAGGTGGG NO: 195 TGGGTGGGTGTGTGTGCGGTGGTGTGATTTGGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTGGTTG NO:196 GTGGGGGGCGGCGGGTGGGGAGCCTGG TGTTCCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTCCCGTTT NO: 197 GAGGGCTTGTCGGACAGATTGCTGGCACGTCACGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTCTTGGTG NO:198 GTGGTGGTGGGTTGGGATGGGTCTTGG GCTGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCCTGTGAG NO: 199 GGGAGGGAGGGTGGGTTTGGCGGTGGCGCAGGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCGTGGTGG NO:200 TGCGTGGGTGGTGGGGGGGGGAGCTGG GTGCCCCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGTGGGT NO: 201 GTTGTGGGGGTGGGTGGTGGGCCCTTCGCCGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTTCCGGTA NO:202 TGTGTGGGTGGGTGGGTGGTGTGGTGG TGTTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTCTCTTCT NO: 203 GTTGTGGGTGGGTGGGTGGTGTGGTGCGTGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCGGCTGGG NO: 204TGGGTTGGGTTAGGGTGGTGTGCGGTG GGTTGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGTTTAGGT NO: 205 GGGCGGGTGGGTGTGCGGTGGGCGGTGTTGAACGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCGGTGATT NO: 206GGGGTTGGGGCCTGGGCGTTTGGGGAC CGCATGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGTTTGGG NO: 207 GTTGGGGCCTGGGAGTTTGGGGAGCAGAGAGGACGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTAACTTGT NO:208 TGGGGTTTGGGGCCTGGGTGTTGGGGT TGTTTCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGGGGTT NO: 209 GGTGGGGGGAGGTGGGTGGGTTATGTGCGCTGGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT NO:210 GTTGTGGGGGTGGGTTGGTGGGCATTG CGTGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGAGTGGG NO: 211 TTCGGTGGTGGTGTGTGGGAGGGTTGGGTACGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGGACAT NO:212 GATTGCACCGTATGAGGTTTAGTCGTTA ATGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCAGTGGGG NO: 213 CCTGGGCGTTGGGGTTTGGGGTGCCTCGTCAGTCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCATGGATTT NO:214 TCGGTGGGTGGGTGGGTTGGTGTGGTG GTGTGCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCTGTGGTTG NO: 215 GTGGGGGGTGGGTGGTGGGAAGGTTCCGGTGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCGGTTGGG NO: 216GTTGGGGCCTGGGTGTTGGGGAGCAGG TAGCACCGTAGTTGAGTCTGAGTGCT SEQ ID DNAGTGACGACTGACATATCTGCGGCCTGG NO: 217 GAGGGTTCGGTGGTGGTGCGAGGGTGGGCAAGCCGTAGTTGAGTCTGAGTGCT SEQ ID DNA ACCTAGTTTGGCTTGCAXAAGTAACYA NO:218 GCACGTGGGCTAG, where X = 5-(amino-1-propenyl)-2′- deoxyuridine; andY = 5-(4- pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACGATCGCCCCYGTCTWTAAGAXCGAA NO: 219 TACTATGGGCTAG, where W= 5-(indole-3-acetamido-1-propenyl)- 2′-deoxyuridine; X= 5-(amino-1-propenyl)-2′- deoxyuridine; and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACCTAGAAAGGCTTAGTGAAGTAAWG NO: 220 ATCAGGGCGGGATC, where W= 5-(indole-3-acetamido-1-propenyl)- 2′-deoxyuridine. SEQ ID DNAACCTAGTTCCCYGTCTAXYAGAXCCGA NO: 221 GXGTATGCCGATC, where X= 5-(amino-1-propenyl)-2′- deoxyuridine; and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACCTAGGCAGTCTTGCCGAATTTACGA NO: 222 GXGGGGAGGGATC, where X= 5-(amino-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACGATCACTGCYCAGCWTYATTAACYA NO: 223 GCYTCGACCCTAG, where W= 5-(indole-3-acetamido-1-propenyl)- 2′-deoxyuridine; and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACGATCTTCCGCCAGCTGYATTXCGAA NO: 224 GXGCGTGAGGATC, where X= 5-(amino-1-propenyl)-2′- deoxyuridine: and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine SEQ ID DNAACCTAGGCGGTCTTXCCGTCGTTACGTC NO: 225 CYCGGCCCCTAG, where X= 5-(amino-1-propenyl)-2′- deoxyuridine; and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACCTAGTTTGGCGTAGCGYATTAAWGG NO: 226 GXGCGGCAGCTAG, where W= 5-(indole-3-acetamido-1-propenyl)- 2′-deoxyuridine; X= 5-(amino-1-propenyl)-2′- deoxyuridine; and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACGATCGCTGACGTXCAXYAGTATGAG NO: 227 GCACGTGGGCTAG, where X= 5-(amino-1-propenyl)-2′- deoxyuridine; and Y = 5-(4-pivaloylbenzamido-1-propenyl)-2′- deoxyuridine. SEQ ID DNAACGGAGAAAGAGAGAGTGTAATTGCTA NO: 228 GCATAACCGCTGC SEQ ID DNAGTAACCACGTTGCCAGACCGAGTCTAC NO: 229 CAGCGATCCTCAG SEQ ID DNATATGCCCAAATCCCTCAAGTCGGCCAG NO: 230 GATACACCACCGT SEQ ID DNAAATCAAAAGGCTCACGCGCGGATTGGT NO: 231 CAACCTTACAACC SEQ ID DNATCGGCCTTCCCAGACCACCGCAATCCCC NO: 232 AGGGAACAGGCA SEQ ID DNACATCACACTGTCAACATACCCAGCCTG NO: 233 GGGAAAGACGAAC SEQ ID DNAAACCCGCATGCCGATCGATGTCGTGCC NO: 234 TCGCTCCACGCTC SEQ ID DNAACCAGGCACCCGACGGACTAACTCATC NO: 235 ACTCAGGCGAGGG

In some cases, an aptamer of the disclosure may have at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity with any aptamer describedherein. For example, an anti-fD aptamer of the disclosure may have atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any aptamerdescribed in Table 1 or Table 2. In some cases, an anti-fD aptamer ofthe disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with SEQ ID NOs:13 or 269. In some cases, an anti-fD aptamer ofthe disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with SEQ ID NOs:165 or 284. In some cases, an anti-fD aptamerof the disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with SEQ ID NOs:166 or 285. In some cases, an anti-fDaptamer of the disclosure may have at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity with SEQ ID NOs:244 or 294. In some cases, ananti-fD aptamer of the disclosure may have at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity with SEQ ID NOs:253 or 303. In some cases,an anti-fD aptamer of the disclosure may have at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity with SEQ ID NOs:256 or 306. In somecases, an anti-fD aptamer of the disclosure may have at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs:262 or 312.

In some cases, an anti-fD aptamer of the disclosure has at least 70%sequence identity with any one of SEQ ID NOs:13, 165, 166, 244, 253,256, 262, 269, 284, 285, 294, 303, 306, and 312. In some cases, ananti-fD aptamer of the disclosure has at least 75% sequence identitywith any one of SEQ ID NOs:13, 165, 166, 244, 253, 256, 262, 269, 284,285, 294, 303, 306, and 312. In some cases, an anti-fD aptamer of thedisclosure has at least 80% sequence identity with any one of SEQ IDNOs:13, 165, 166, 244, 253, 256, 262, 269, 284, 285, 294, 303, 306, and312. In some cases, an anti-fD aptamer of the disclosure has at least85% sequence identity with any one of SEQ ID NOs:13, 165, 166, 244, 253,256, 262, 269, 284, 285, 294, 303, 306, and 312. In some cases, ananti-fD aptamer of the disclosure has at least 90% sequence identitywith any one of SEQ ID NOs:13, 165, 166, 244, 253, 256, 262, 269, 284,285, 294, 303, 306, and 312. In some cases, an anti-fD aptamer of thedisclosure has at least 95% sequence identity with any one of SEQ IDNOs:13, 165, 166, 244, 253, 256, 262, 269, 284, 285, 294, 303, 306, and312.

In some cases, an aptamer of the disclosure may have a primarynucleotide sequence that has at least 86%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence homology with any aptamer described herein. For example, ananti-fD aptamer of the disclosure may have a primary nucleotide sequencethat has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology withany aptamer described in Table 1 or Table 2. In some cases, an aptamerof the disclosure may have a primary nucleotide sequence that has atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ IDNOs:13 or 269.

In some cases, an aptamer of the disclosure may have a primarynucleotide sequence that shares at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17; atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 30, at least 31, at least 32, at least 33, at least34, at least 35, at least 36, at least 37, at least 38, at least 39, orat least 40 contiguous nucleotides with a nucleotide sequence describedin Table 1 or Table 2. In some cases, an aptamer of the disclosure mayhave a primary nucleotide sequence that shares at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, orat least 39 contiguous nucleotides with SEQ ID NOs:13 or 269. In somecases, an aptamer of the disclosure may have a primary nucleotidesequence that shares at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, atleast 30, at least 31, at least 32, at least 33, at least 34, at least35, at least 36, at least 37, at least 38, or at least 39 contiguousnucleotides with SEQ ID NOs:165 or 284. In some cases, an aptamer of thedisclosure may have a primary nucleotide sequence that shares at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, or at least 39 contiguous nucleotides with SEQ ID NOs:166 or285. In some cases, an aptamer of the disclosure may have a primarynucleotide sequence that shares at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 30, at least 31, at least 32, at least 33, at least34, at least 35, at least 36, at least 37, at least 38, or at least 39contiguous nucleotides with SEQ ID NOs:244 or 294. In some cases, anaptamer of the disclosure may have a primary nucleotide sequence thatshares at least 10, at least 11, at least 12, at least 13, at least 14,at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, at least 27, at least 28, at least 29, at least 30, atleast 31, at least 32, at least 33, at least 34, at least 35, at least36, at least 37, at least 38, or at least 39 contiguous nucleotides withSEQ ID NOs:253 or 303. In some cases, an aptamer of the disclosure mayhave a primary nucleotide sequence that shares at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, orat least 39 contiguous nucleotides with SEQ ID NOs:256 or 306. In somecases, an aptamer of the disclosure may have a primary nucleotidesequence that shares at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, atleast 30, at least 31, at least 32, at least 33, at least 34, at least35, at least 36, at least 37, at least 38, or at least 39 contiguousnucleotides with SEQ ID NOs:262 or 312.

In such cases where specific nucleotide modifications have been recited,it should be understood that any number and type of nucleotidemodifications may be substituted. For example, 2′OMeG may be substitutedfor 2′FG. Non-limiting examples of nucleotide modifications have beenprovided herein. In some instances, all of the nucleotides of an aptamerare modified. In some instances, at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the nucleotides of an aptamer of the disclosure maybe modified.

Aptamers of the disclosure may have a stem-loop secondary structurecomprising at least one stem and at least one loop. FIG. 1 depicts anexemplary consensus nucleotide sequence of aptamers in relation to thestructural elements of the aptamer. Although particular sequences andstructural elements are described in FIG. 1, it should be understoodthat FIG. 1 is meant only as an example of a graphical representation ofa family of anti-fD aptamers. Other structurally-related families ofanti-fD aptamers are described herein. In some cases, aptamers of thedisclosure may be structurally related to an aptamer of FIG. 1, but maytolerate some variation in size or nucleic acid sequence while stillretaining anti-fD activity.

In various aspects, an aptamer of the disclosure may comprise a nucleicacid sequence that selectively blocks or occludes the active site of fD.The anti-fD aptamer may have a stem-loop secondary structure comprisingat least one stem and at least one loop. In some cases, the anti-fDaptamer has a nucleic acid sequence comprising from 30 to 90nucleotides. For example, the anti-fD aptamer may have a nucleic acidsequence comprising 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 nucleotides.

In various aspects, an aptamer of the disclosure may comprise a nucleicacid sequence that selectively binds to fD. The anti-fD aptamer may havea stem-loop secondary structure comprising at least one stem and atleast one loop. In some cases, the anti-fD aptamer comprises at leastone modified nucleotide.

In various aspects, an aptamer of the disclosure may comprise a nucleicacid sequence that selectively binds to fD. The anti-fD aptamer may havea stem-loop secondary structure comprising at least one stem and atleast one loop. In some cases, the anti-fD aptamer does not include anucleic acid sequence of any one of SEQ ID NOs:228-235. In someinstances, the anti-fD aptamer does not include a nucleic acid sequenceof any one of SEQ ID NOs:1-3, 168-227. In some cases, the anti-fDaptamer does not include a nucleic acid sequence of any one of SEQ IDNOs:168-227.

In various aspects, an aptamer of the disclosure may comprise a nucleicacid sequence that selectively blocks an active site of fD. The anti-fDaptamer may have a secondary structure having exactly three loops. Insome instances, the anti-fD aptamer may have exactly two stems.

In various aspects, an aptamer of the disclosure may comprise a nucleicacid sequence that selectively blocks an activity of fD. The anti-fDaptamer may have a secondary structure having less than four loops. Forexample, the anti-fD aptamer may have three loops, two loops, or oneloop. In some cases, a secondary structure of the anti-fD aptamer aspredicted by M-fold contains less than a total of 15 unpaired residuesat either terminus. For example, a secondary structure of the anti-fDaptamer as predicted by M-fold contains 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1, or 0 total unpaired residues at either terminus. Insome cases, a secondary structure of the anti-fD aptamer as predicted byM-fold has less 4 loops.

In some cases, a secondary structure of the anti-fD aptamer as definedby comparative sequence analysis and multiple sequence alignmentcontains less than a total of 15 unpaired residues at the 5′ terminus.For example, a secondary structure of the anti-fD aptamer as defined bycomparative sequence analysis and multiple sequence alignment maycontain 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 unpairednucleotide residues at the 5′ terminus. In some cases, a secondarystructure of the anti-fD aptamer as defined by comparative sequenceanalysis and multiple sequence alignment contains less than 15 unpairedresidues at the 3′ terminus. For example, a secondary structure of theanti-fD aptamer as defined by comparative sequence analysis and multiplesequence alignment may contain 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, 1, or 0 unpaired nucleotide residues at the 3′ terminus. In somecases, a secondary structure of the anti-fD aptamer as defined bycomparative sequence analysis and multiple sequence alignment containsless than 30 total unpaired nucleotide residues at the 5′ and 3′termini. For example, a secondary structure of the anti-fD aptamer asdefined by comparative sequence analysis and multiple sequence alignmentmay contain 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 total unpairednucleotide residues at the 5′ and 3′ termini. In some cases, a secondarystructure of the anti-fD aptamer as defined by comparative sequenceanalysis and multiple sequence alignment has less 4 loops.

In various aspects, an aptamer of the disclosure may comprise a nucleicacid sequence that selectively binds to fD. The anti-fD aptamer may havea stem-loop secondary structure that comprises at least one stem and atleast one loop. In some cases, the aptamer is an RNA aptamer or amodified RNA aptamer. In various aspects, an aptamer of the disclosuremay be single-stranded, for example, may have no more than one nucleicacid strand. In other aspects, an aptamer of the disclosure may havemore than one nucleic strands, for example, may have two or more nucleicstrands.

In some cases, the anti-fD aptamer may have up to two stems, forexample, the anti-fD aptamer may have one stem, or two stems. In somecases, each of the stems may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 base pairs. In somecases, each of the stems may have less than 20, less than 19, less than18, less than 17, less than 16, less than 15, less than 14, less than13, less than 12, less than 11, less than 10, less than 9, less than 8,less than 7, less than 6, less than 5, less than 4, less than 3, or lessthan 2 base pairs. In some instances, no more than one of the stems hasmore than 20 base pairs. In some cases, one or more of the stems mayhave one or more mismatched base pairs.

In some cases, the anti-fD aptamer may have up to three loops, forexample, the anti-fD aptamer may have three loops, two loops, or oneloop. In some cases, each of the loops may have up to 25 nucleotides.For example, each loop may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Insome cases, at least one loop may have more than 1, more than 2, morethan 3, more than 4, more than 5, more than 6, more than 7, more than 8,more than 9, or more than 10 nucleotides.

In particular aspects, aptamers of the disclosure may have a stem-loopsecondary structure that includes, in a 5′ to 3′ direction, a first stem(S1), a first loop (L1), a second stem (S2), a second loop (L2), and athird loop (L3). As demonstrated in FIG. 1, the first loop (L1) may beconnected to the 3′ terminal end of the first stem (S1) and the 5′terminal end of the second stem (S2). The second stem (S2) may beconnected to the 3′ terminal end of the first loop (L1) and the 5′terminal end of the second loop (L2). The second loop (L2) may beconnected to the 3′ terminal end of the second stem (S2) and the 5′terminal end of the complementary region of the second stem (S2). Thecomplementary region of the second stem (S2) may be connected to the 3′terminal end of the second loop (L2) and the 5′ terminal end of thethird loop (L3). The third loop (L3) may be connected to the 3′ terminalend of the complementary region of the second stem (S2) and the 5′terminal end of the complementary region of the first stem (S1). In somecases, the first loop (L1) may comprise fewer nucleotides than thesecond loop (L2). In some cases, the third loop (L3) is connected to thefirst stem (S1) (e.g., connected to the 5′ terminal end of thecomplementary region of the first stem (S1)).

In some aspects, the first loop (also referred to as L1) may have from 1to 10 nucleotides. For example, the first loop (L1) may have 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides. In particular aspects, the first loop(L1) may have from 3 to 5 nucleotides. For example, the first loop (L1)may have 3, 4 or 5 nucleotides. In some cases, the first loop (L1) has 3or less nucleotides. For example, the first loop (L1) may have 1, 2, or3 nucleotides. In some cases, the first loop (L1) has 3 or morenucleotides. For example, the first loop (L1) may have 3, 4, 5, 6, 7, 8,9, or 10 nucleotides. In some cases, the first loop (L1) has greaterthan 4 nucleotides. In some cases, the first loop (L1) has greater than5 nucleotides. In some cases, the first loop (L1) has greater than 6nucleotides. In some cases, the first loop (L1) has greater than 7nucleotides. In some cases, the first loop can contain one or morenon-nucleotidyl spacers in place of nucleotides (e.g., a 3-carbonspacer, a 6-carbon spacer, a 9-carbon spacer, or an 18-atom spacer (suchas a hexaethylene glycol spacer). In a non-limiting example, the firstloop (L1) may comprise a nucleic acid sequence of 5′-DUG-3′, where D isA, G, or U.

In some aspects, the second loop (L2) may have from 2 to 15 nucleotides.For example, the second loop (L2) may have 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 nucleotides. In a particular example, the secondloop (L2) may have at least 6 nucleotides. In another particularexample, the second loop (L2) may have at least 8 nucleotides. Inanother particular example, the second loop (L2) may have exactly 10nucleotides. In yet another particular example, the second loop (L2) mayhave 10 or 11 nucleotides. In some cases, the second loop (L2) has 7 ormore nucleotides. For example, the second loop (L2) may have 7, 8, 9,10, 11, 12, 13, 14, or 15 nucleotides. In some cases, the second loop(L2) has 10 or more nucleotides. For example, the second loop (L2) mayhave 10, 11, 12, 13, 14, or 15 nucleotides. In some cases, the secondloop (L2) has 10 or less nucleotides. For example, the second loop (L2)may have 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. In some cases, thesecond loop (L2) has greater than 4 nucleotides. In some cases, thesecond loop (L2) has greater than 5 nucleotides. In some cases, thesecond loop (L2) has greater than 6 nucleotides. In some cases, thesecond loop (L2) has greater than 7 nucleotides. In some cases, thesecond loop can contain one or more non-nucleotidyl spacers in place ofnucleotides (e.g., a 3-carbon spacer, a 6-carbon spacer, a 9-carbonspacer, or an 18-atom spacer (such as a hexaethylene glycol spacer). Ina non-limiting example, the second loop (L2) may comprise a nucleic acidsequence of 5′-DWWVGCBHWG-3′ (SEQ ID NO:319), where D is A, G, or U; Wis A or U; V is A, C, or G; B is C, G, or U; and H is A, C, or U. Insome cases, the second loop (L2) comprises a nucleic acid sequencehaving a U at nucleotide position 2 of the second loop (L2), atnucleotide position 3 of the second loop (L2), or both. In some cases,the first loop (L1) has fewer nucleotides than the second loop (L2).

In some aspects, the third loop (L3) may have from 2 to 10 nucleotides.For example, the third loop (L3) may have 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides. In a particular example, the third loop (L3) may have atleast 6 nucleotides. In some cases, the third loop (L3) has exactly 6nucleotides. In other cases, the third loop (L3) may have from 6 to 8nucleotides, for example, 6, 7, or 8 nucleotides. In some cases, thethird loop (L3) has 6 or more nucleotides, for example, 6, 7, 8, 9, or10 nucleotides. In some cases, the third loop (L3) has 6 or lessnucleotides, for example, 6, 5, 4, 3, or 2 nucleotides. In some cases,the third loop (L3) has greater than 4 nucleotides. In some cases, thethird loop (L3) has greater than 5 nucleotides. In some cases, the thirdloop (L3) has greater than 6 nucleotides. In some cases, the third loop(L3) has greater than 7 nucleotides. In some cases, the third loop cancontain one or more non-nucleotidyl spacers in place of nucleotides(e.g., a 3-carbon spacer, a 6-carbon spacer, a 9-carbon spacer, or an18-atom spacer (such as a hexaethylene glycol spacer). In some examples,the third loop (L3) has a nucleic acid sequence comprising 5′-AAGUKN-3′,where K is G or U; and N is A, G, C, or U. In some cases, the third loop(L3) is connected to the first stem (S1). In some cases, the third loop(L3) has at least 4 nucleotides and up to 2 non-nucleotidyl spacers.

In some aspects, the first stem (S1) may have from 2 to 10 base pairs.For example, the first stem (S1) may have 2, 3, 4, 5, 6, 7, 8, 9, or 10base pairs. In a particular example, the first stem (S1) may have from 3to 8 base pairs, for example, 3, 4, 5, 6, 7, or 8 base pairs. In somecases, the first stem (S1) has 10 or less base pairs, for example, 10,9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. In some cases, the first stem(S1) has 5 or less base pairs, for example, 5, 4, 3, or 2 base pairs. Insome cases, the first stem (S1) may include one or more mismatched basepairs.

In some aspects, the second stem (S2) may have from 2 to 10 base pairs.For example, the second stem (S2) may have 2, 3, 4, 5, 6, 7, 8, 9, or 10base pairs. In a particular example, the second stem (S2) may have 4 or5 base pairs. In some cases, the second stem (S2) has 10 or less basepairs, for example, 10, 9, 8, 7, 6, 5, 4, 3, or 2 base pairs. In somecases, the second stem (S2) has 5 or less base pairs, for example, 5, 4,3, or 2 base pairs. In some cases, the second stem (S2) may comprise aterminal U-G base pair, adjacent to the second loop (L2).

In various aspects, an aptamer of the disclosure is conjugated to apolyethylene glycol (PEG) molecule at the 5′ end of the aptamer. Variousnon-limiting examples of PEG molecules suitable for use with an aptamerof the disclosure are provided throughout. In some cases, an aptamer ofthe disclosure is conjugated to a PEG molecule having a molecular weightof 80 kDa or less (e.g., 40 kDa).

In some cases, an aptamer of this disclosure may include: (1) a terminalstem (S1); (2) an asymmetric internal loop containing a 5′ side (L1) anda 3′ side (L3); (3) a second stem (S2); and (4) a terminal loop (L2). Insome cases, an aptamer of this disclosure may contain any of thefollowing: (1) a terminal stem (S1) having 3-8 base pairs; (2) anasymmetric internal loop, the 5′ side of which (L1) ranging from 3-5nucleotides; (3) a second stem (S2) having 4-5 base pairs; (4) aterminal loop (L2) that is 10 to 11 nucleotides in length; and (5) a 3′side of the asymmetric internal loop (L3) having 6-8 nucleotides.

In some cases, an aptamer of the disclosure comprises a nucleic acidsequence that selectively binds to complement factor D (fD) and has astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop. In one aspect, the third loop maycomprise 6 or more nucleotides, non-nucleotidyl spacers, or acombination thereof, and the first loop may have fewer nucleotides thansaid second loop. In another aspect, the third loop may comprise 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andthe second loop may comprise more than 5 nucleotides, non-nucleotidylspacers, or a combination thereof. In another aspect, the third loop maycomprise 6 or more nucleotides, non-nucleotidyl spacers, or acombination thereof, and the third loop may be adjacent to the firststem. In yet another aspect, the third loop may comprise 6 or morenucleotides, non-nucleotidyl spacers, or a combination thereof, and thefirst base-paired stem may have no more than 5 base pairs. In yetanother aspect, the second loop may comprise 7 or more nucleotides,non-nucleotidyl spacers, or a combination thereof, the first base-pairedstem may have no more than 5 base pairs, and the second base-paired stemmay comprise more than 2 base pairs. In another aspect, the secondbase-paired stem may comprise a terminal U-G base pair adjacent to thesecond loop. In another aspect, the first loop may comprise a nucleicacid sequence of 5′-DUG-3′, where D is A, G, or U. In another aspect,the third loop may comprise a nucleic acid sequence comprising5′-AAGUKN-3′, where K is G or U; and N is A, G, C, or U. In yet anotheraspect, the second loop may comprise a nucleic acid sequence of5′-DWWVGCBHWG-3′ (SEQ ID NO:319), where D is A, G, or U; W is A or U; Vis A, C, or G; B is C, G, or U; and H is A, C, or U. In another aspect,the second loop may comprise a nucleic acid sequence having a U atnucleotide position 2, nucleotide position 3, or both. In anotheraspect, the second base-paired stem may comprise a terminal C-G basepair adjacent to the second loop. In some cases, the first base-pairedstem is adjacent to the first loop. In some cases, the secondbase-paired stem is adjacent to the first loop, the second loop, and thethird loop. In some cases, the first base-paired stem is adjacent to thefirst loop and the second base-paired stem is adjacent to the firstloop, the second loop, and the third loop.

In some cases, an aptamer of the disclosure comprises a nucleic acidsequence that selectively binds to complement factor D (fD) and has astem-loop secondary structure comprising a base-paired terminal stem; anasymmetric internal loop; an internal base-paired stem; and exactly oneterminal loop. In some aspects, the terminal loop may comprise more than4 nucleotides, non-nucleotidyl spacers, or a combination thereof, andthe asymmetric internal loop may be adjacent to exactly 2 base-pairedstems.

In some cases, an aptamer of the disclosure comprises a nucleic acidsequence that selectively binds to complement factor D (ID) and has astem-loop secondary structure comprising exactly one terminalbase-paired stem; exactly one asymmetric internal loop comprising, froma 5′ to 3′ direction, a first loop and a second loop; exactly oneinternal base-paired stem; and exactly one terminal loop. In someaspects, the first loop of the asymmetric internal loop may have fewernucleotides than the terminal loop. In another aspect, the exactly oneterminal loop may comprise more than 4 nucleotides, non-nucleotidylspacers, or a combination thereof. In another aspect, the second loopmay comprise 6 or more nucleotides, non-nucleotidyl spacers, or acombination thereof. In yet another aspect, the exactly one terminalloop may comprise 7 or more nucleotides, non-nucleotidyl spacers, or acombination thereof.

Anti-fD Compositions

fD is a component of the alternative complement pathway and is believedto be involved in the pathogenesis of AMD and other ocular disorders. fDis unique among serine proteases in that it does not require cleavage ofa zymogen for expression of proteolytic activity. Rather, fD requires aconformational change that is believed to be induced by the complex C3bBresulting in a reversible reorientation of the catalytic center andsubstrate binding site of fD. fD is primarily produced by adipocytes andis systemically available in serum at low levels. fD contains aself-inhibitory loop that prevents catalytic activity of fD. Binding ofthe C3bB complex to fD displaces the self-inhibitory loop and fD cleavesC3bB to form the C3 convertase C3bBb. The catalytic activity of fD onlyoccurs in the context of complexed fB; fD does not cleave uncomplexedfB. The complex of fD, fB, and C3b forms an amplification loop of thealternative complement pathway of which fD is the rate-limited enzyme.

In some aspects, the methods and compositions described herein involveinhibition of fD, resulting in inhibition of the amplification step ofthe alternative complement pathway. The anti-fD compositions herein mayinvolve the use of one or more anti-fD aptamers for the treatment ofocular diseases. In some cases, the ocular disease is maculardegeneration. In some cases, macular degeneration is age-related maculardegeneration. In some cases, age-related macular degeneration is dryage-related macular degeneration. In some cases, dry age-related maculardegeneration is advanced dry age-related macular degeneration (i.e.,geographic atrophy). In some cases, age-related macular degeneration iswet age-related macular degeneration. In some cases, maculardegeneration is Stargardt disease or Stargardt-like disease.

Anti-fD Inhibitors

The anti-fD compositions disclosed herein may be designed to bind tospecific regions of fD with high specificity and affinity. Thecompositions may bind to fD in such a way as to inhibit, either directlyor indirectly, the catalytic activity of the enzyme. In some cases, theanti-fD aptamers can bind to the active site (e.g., the catalytic cleft)of fD and directly inhibit the catalytic activity of fD. In thisexample, the aptamer may be designed to target the active site (e.g.,the catalytic cleft) of fD. In other cases, the anti-fD aptamers canbind to a region at or near the active site, such that binding of theaptamer occludes or blocks the substrate from accessing the active site.When the aptamer is bound to the active site of fD, it can prevent thesubstrate (e.g., C3bB) from accessing the active site. In some cases,the anti-fD aptamer can bind to an exosite of fD and indirectly inhibitthe catalytic activity of fD by e.g., preventing the binding of C3bB. Insome cases, the exosite may be remote from the catalytic site. In othercases, there may be some overlap with the catalytic site. In some casesthe anti-fD aptamer can bind to the self-inhibitory loop of fD toprevent displacement of the self-inhibitory loop and thus, preventactivation of fD.

Amino acid residues of fD may be referenced according to thechymotrypsin numbering scheme and this numbering system is usedthroughout the disclosure to refer to specific amino acid residues offD. Chymotrypsin numbering scheme for fD may be as depicted in FIG. 6(SEQ ID NO:9)(chymotrypsin numbering displayed above amino acid sequenceand fD numbering scheme below amino acid sequence).

Anti-fD aptamers as described herein can modulate or inhibit theactivity of fD or a fD variant thereof. A fD variant as used hereinencompasses variants that perform essentially the same function as fD. AfD variant includes essentially the same structure as fD and in somecases includes at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity tothe amino acid sequence (shown above) of the fD protein.

In certain embodiments of the disclosure, methods are provided for theidentification of fD aptamers that specifically bind to epitopes of fD.These methods may be utilized, for example, to determine the bindingsite and/or the mechanism of action of the aptamer.

In one instance, methods are provided for testing a fD aptamer inalternative complement dependent hemolysis of red blood cells. Humanserum that is rendered deficient in the classical complement pathway bydepleting C1q may be dependent on alternative complement activity tolyse rabbit red blood cells, an activity that may be dependent on fD(Katschke, W u, Ganesan, et al. (2012) Inhibiting alternative pathwaycomplement activation by targeting the Factor D exosite. J. Biol. Chem.287, 12886-12892). In some cases, the fD aptamers disclosed herein mayinhibit alternative complement dependent hemolysis of red blood cells(see Example 2).

In another instance, methods are provided for testing a fD aptamer in fDesterase activity assays (see Example 3). Cleavage of a modified peptidesubstrate of fD, Z-lys-S-Bzl, may be monitored by the cleaved productreducing 5,5′-Dithiobis(2-nitrobenzoic acid). FD may have a lowercatalytic rate than other complement proteases when using peptidethioester substrates, and one such substrate Z-lys-SBzl was found to becleaved by fD and useful as a synthetic substrate (fD is called proteinD in Kam, McRae et al. (1987) Human complement proteins D, C2, and B. J.Biol. Chem. 262, 3444-3451). In some cases, a molecule that binds fD mayblock catalytic activity by binding in the catalytic cleft to stericallyprevent access of the peptide substrate to the catalytic residues of fD(Katschke, W u, Ganesan, et al. (2012) Inhibiting alternative pathwaycomplement activation by targeting the Factor D exosite. J. Biol. Chem.287, 12886-12892). In other cases, a molecule that binds fD may blockcatalytic activity by occluding access of the substrate to the activesite. In yet other cases, a molecule that binds fD may block catalyticactivity by an allosteric mechanism that induces structural changes inthe enzyme. In yet other cases, a molecule that binds fD may bind to thefD exosite region to sterically inhibit binding of the physiologicsubstrate protein C3bB, but not of the synthetic modified peptidesubstrate Z-Lys-SBzl (Katschke, Wu, Ganesan, et al. (2012) Inhibitingalternative pathway complement activation by targeting the Factor Dexosite. J. Biol. Chem. 287, 12886-12892). In some instances, where amolecule inhibits fD binding and proteolytic cleavage of fB but notZ-Lys-SBzl, the binding may be similar to how anti-factor D FAb antibodyfragment binds to the exosite and induces a subtle conformational changethat increases fD cleaving Z-Lys-S-Bzl (Katschke, Wu, Ganesan, et al.(2012) Inhibiting alternative pathway complement activation by targetingthe Factor D exosite. J. Biol. Chem. 287, 12886-12892).

In another instance, methods are provided for testing a fD aptamer in areconstituted biochemical fD activity assay which is composed ofpurified proteins fD, fB, and C3b (see Example 4). When fD binds to thecomplex of fB and C3b (C3bB), fB is cleaved by fD into fragments Ba andBb (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathwaycomplement activation by targeting the Factor D exosite. J. Biol. Chem.287, 12886-12892). The activity of fD can be monitored by the rate of fBcleavage and Ba fragment production using an ELISA that uses an antibodythat specifically binds Ba (Quidel, A033), or by other means known inthe art to measure Ba levels. In some cases, the concentrations of fBand C3b are equal so they form a 1:1 complex which can then bind fD andallow enzymatically active fD to cleave fB to fragments Ba and Bb. Insome cases, the fB:C3b complex is present in 4-fold excess of fD. Inother cases, the concentrations of fD and/or C3bB are varied in such amanner as to measure enzymatic constants, including, but not limited tok_(cat), K_(m) and k_(cat)/K_(m).

In yet another instance, methods are provided for the identification offD binding to C3bB in complex (see Example 5). FD is the rate-limitingenzyme in the alternative complement pathway, and converts theproconvertases C3bB and C3b₂B to form the active C3 convertase C3bBb orthe active C5 convertase C3b₂Bb (Katschke et al 2012). For surfaceplasmon resonance (SPR) to detect fD in a stable complex with fB,catalytically inactive fD (S195A) may be used so that it does not cleavethe fB upon binding to the fB:C3b complex (Katschke, Wu, Ganesan, et al.(2012) Inhibiting alternative pathway complement activation by targetingthe Factor D exosite. J. Biol. Chem. 287, 12886-12892). When C3b isamine-coupled to a CM5 chip, SPR may detect binding of fB as increasedmass, and binding of fD to the C3b:fB complex as a further increase inmass. In one aspect, the fD binding compounds are aptamers that bind fDand prevent fD binding to fB:C3b as determined by a reduced massdetected by SPR.

In some cases, a cell model of Stargardt disease may be used to detectactivity of anti-fD aptamers (see Example 6). Retinal pigment epithelial(RPE) cells may undergo cell death early during the progress ofStargardt disease, and evidence points toward the involvement of thealternative complement pathway (AP) in RPE cell death (Berchuck, Yang,et al (2013) All-trans-retinal (atRal) sensitizes human RPE cells toalternative complement pathway-induced cell death. Invest Ophthalmol VisSci 54, 2669-2677). ARPE-19 cells are a spontaneously arising RPE cellline derived from the normal eyes of a 19-year-old male. The ARPE-19cell line, established using the cuboidal basal cell layer cultured inspecific culture media, expresses the RPE-specific markers cellularretinaldehyde binding protein and RPE-65. Stargardt disease is ahereditary juvenile macular degeneration that occurs in patients withhomozygous mutations in the ABCA4 genes, which encode a protein that isresponsible for removal of bisretinoid fluorophores, which can includeN-retinylidene-N-retinyethanolamine (A2E), all-trans-retinal and relatedphoto-oxidation products of vitamin A aldehyde which together constitutelipofuscin from photoreceptor cells (Molday (2007) ATP-binding cassettetransporter ABCA4: molecular properties and role in vision and maculardegeneration. J. Bioenerg Biomembr 39, 507-517). An ABCA4 and RDH8 mousemodel of Stargardt disease presents with retinal pathology caused byaccumulated atRal, and ABCA4 mutations are present in 16% of AMDpatients, suggesting that elevated atRal may contribute to Stargardtdisease and AMD disease progression (Berchuck et al 2013).Mechanistically, atRal decreased expression of CD46 and CD59 on RPEcells in vitro, which increased susceptibility to cell lysis mediated byalternative complement in response to anti-RPE antibody binding to theRPE cell membranes (Berchuck et al 2013). In some cases, the disclosureprovides for the identification of fD inhibitors that inhibitalternative complement-mediated lysis of human retinal pigmentedepithelial cells.

The anti-fD aptamers as disclosed herein, in some cases, may bind to theregion of fD that includes the active site cleft. Upon activation bybinding to C3bB, fD exhibits serine protease activity towards fB.Activation of fD by substrate binding is a two-step process: first, fDbinds to fB in the open C3bB configuration at the Von Willebrand factortype-A (VWA)-serine protease (SP) interface of fB, interacting mainlyvia its exosite residues within loops 145-149, 169-173, 185-188 and220-224. Binding of fD to C3bB causes the self-inhibitory loop of fD tobe displaced from the active site cleft. The global architecture of fDis comprised of two anti-parallel beta barrel domains, each composed ofsix or seven beta strands that have the same topology in both domains.The beta-strands are connected by 14 turns/loops and three short alphahelices. The active site cleft is located within the loop formed betweenthe two beta barrels, and encompasses structural elements includinghelix 1, loop 7 and beta-strand 7, loop 11 and beta-strand 11,beta-strand 12, loop 13 and beta-strand 13 (Jing et. al. 1998). Aptamerswhich bind the active site cleft could recognize any portion of thealpha helices, loops and beta strands which comprise the portion of fDwithin which the active site cleft resides, and by binding to thisregion, may prevent access to the active site cleft. Such residuesinclude the catalytic triad, His57, Asp102 and Ser195, the oxyanion holeincluding the backbone amine of residue 193 and Ser195, the residueslinking the catalytic triad to the oxyanion hole via a salt bridgeincluding residue 16, 194 and Ser195, the S1 pocket, including residues189-192, 214-216, and 224-228, as well as other elements of thespecificity pocket including those residues comprising the S2, S3, S4and Sn pockets. In particular, such aptamers would prevent interactionof P2-Pn residues of fB with specificity pockets S2-Sn of fD. In somecases, the aptamers as described herein specifically bind to the activesite cleft or a region comprising the active site cleft of fD. Aptamersthat are said to bind to the active site cleft or a region comprisingthe active site cleft may include any aptamers that bind to one or moreof the regions including the catalytic triad (His57, Asp102 and Ser195);the oxyanion hole including the backbone amine of residue 193 andSer195; the residues linking the catalytic triad to the oxyanion holevia a salt bridge including residue 16, 194 and Ser195; the S1 pocket,including residues 189-192, 214-216, and 224-228; as well as otherelements of the specificity pocket including those residues comprisingthe S2, S3, S4 and Sn pockets.

Such fD inhibitors may inhibit alternative complement dependenthemolysis of red blood cells, may inhibit esterase activity of fDagainst thioester substrates of fD such as Z-Lys-S-Bzl, and may inhibitfB cleavage in the C3bB complex by fD. In esterase assays, suchinhibitors may reduce k_(cat) and increase K_(m) of fD, with the primaryeffect decreasing k_(cat) and decreasing k_(cat)/K_(m) (Hedstrom). Incomplete biochemical assays, such inhibitors may decrease k_(cat) andincrease K_(m), with a primary effect decreasing k_(cat) and decreasingk_(cat)/K_(m). Such inhibitors may not prevent formation of theenzyme-substrate complex (fD-C3bB complex) as assessed in enzymaticassays or enzyme-substrate assembly assays, such as surface plasmonresonance (SPR) assays described in Forneris et. al. or Katschke et.al., or similar E-S assembly assays assessed by ELISA or similar assays.Alternatively, such inhibitors may additionally prevent formation of theenzyme-substrate complex (fD-C3bB complex) as assessed in enzymaticassays or enzyme-substrate assembly assays, such as surface plasmonresonance (SPR) assays described in Forneris et. al. or Katschke et.al., or similar E-S assembly assays assessed by ELISA or similar assays.

In some cases, an anti-fD aptamer as disclosed herein may bind to fDwith a K_(d) of less than about 50 nM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 25 nM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 10 nM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 5 nM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 1 nM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 500 pM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 50 pM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay. In somecases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 5 pM and may inhibit at least 85% of fDactivity in an alternative complement dependent hemolysis assay.

In some cases, an anti-fD aptamer as disclosed herein may bind to fDwith a K_(d) of less than about 50 nM and may inhibit at least 85% of fDactivity in a fD convertase assay. In some cases, an anti-fD aptamer asdisclosed herein may bind to fD with a K_(d) of less than about 25 nMand may inhibit at least 85% of fD activity in a fD convertase assay. Insome cases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 10 nM and may inhibit at least 85% of fDactivity in a fD convertase assay. In some cases, an anti-fD aptamer asdisclosed herein may bind to fD with a K_(d) of less than about 5 nM andmay inhibit at least 85% of fD activity in a fD convertase assay. Insome cases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 1 nM and may inhibit at least 85% of fDactivity in a fD convertase assay. In some cases, an anti-fD aptamer asdisclosed herein may bind to fD with a K_(d) of less than about 500 pMand may inhibit at least 85% of fD activity in a fD convertase assay. Insome cases, an anti-fD aptamer as disclosed herein may bind to fD with aK_(d) of less than about 50 pM and may inhibit at least 85% of fDactivity in a fD convertase assay. In some cases, an anti-fD aptamer asdisclosed herein may bind to fD with a K_(d) of less than about 5 pM andmay inhibit at least 85% of fD activity in a fD convertase assay.

In some cases, an anti-fD aptamer as disclosed herein may bind to fDwith a K_(d) of less than about 50 nM and inhibit fD activity in anesterase activity assay. In some cases, an anti-fD aptamer as disclosedherein may bind to fD with a K_(d) of less than about 25 nM and mayinhibit fD activity in an esterase activity assay. In some cases, ananti-fD aptamer as disclosed herein may bind to fD with a K_(d) of lessthan about 10 nM and may inhibit fD activity in an esterase activityassay. In some cases, an anti-fD aptamer as disclosed herein may bind tofD with a K_(d) of less than about 5 nM and may inhibit fD activity inan esterase activity assay. In some cases, an anti-fD aptamer asdisclosed herein may bind to fD with a K_(d) of less than about 1 nM andmay inhibit fD activity in an esterase activity assay. In some cases, ananti-fD aptamer as disclosed herein may bind to fD with a K_(d) of lessthan about 500 pM and may inhibit fD activity in an esterase activityassay. In some cases, an anti-fD aptamer as disclosed herein may bind tofD with a K_(d) of less than about 50 pM and may inhibit fD activity inan esterase activity assay. In some cases, an anti-fD aptamer asdisclosed herein may bind to fD with a K_(d) of less than about 5 pM andmay inhibit fD activity in an esterase activity assay.

The anti-fD aptamers as disclosed herein, in some cases, may bind to theregion of fD that includes the self-inhibitory loop (residues 212-218)and regions adjacent to the self-inhibitory loop, so as to stabilize theself-inhibited state of fD. Mature fD maintains a self-inhibited statethrough a set of conformations in the free fD state including theconformation of residues 212-218, which may be referred to as theself-inhibitory loop of fD. These residues may comprise portions of thepolypeptide binding site as well as the S1 specificity pocket of fD. Inthe inactive state of fD, this loop is in an elevated conformation andforms specific bonds with key components of the catalytic triad and S1specificity pocket, rendering fD inactive. In some cases, the anti-fDcompounds of the disclosure are designed to target the self-inhibitoryloop of fD to prevent the activation of fD. For example, the anti-fDcompounds may bind to the self-inhibitory loop or to regions around theself-inhibitory loop to prevent displacement of the self-inhibitory loopfrom the active site cleft. In some cases, the anti-fD compounds may bedesigned to target residues 212-218 of fD. In cases where anti-fDaptamers bind to a region comprising one or more of amino acid residues212-218 of fD, it may be said that such anti-fD aptamers bind to theself-inhibitory loop or a portion thereof of fD.

Such fD inhibitors may inhibit alternative complement dependenthemolysis of red blood cells, may inhibit esterase activity of fDagainst thioester substrates of fD such as Z-Lys-S-Bzl, and may inhibitfB cleavage in the C3bB complex by fD. In esterase assays, suchinhibitors may reduce k_(cat) and increase K_(m) of fD, with the primaryeffect decreasing k_(cat) and decreasing k_(cat)/K_(m). In completebiochemical assays, such inhibitors may decrease k_(w) and increaseK_(m), with a primary effect decreasing k_(cat) and decreasingk_(cat)/K_(m). Such inhibitors may not prevent formation of theenzyme-substrate complex (fD-C3bB complex) as assessed in enzymaticassays or enzyme-substrate assembly assays, such as surface plasmonresonance (SPR) assays described in Forneris et. al. or Katschke et.al., or similar E-S assembly assays assessed by ELISA or similar assays.Alternatively, such inhibitors may also prevent formation of theenzyme-substrate complex (fD-C3bB complex) as assessed in enzymaticassays or enzyme-substrate assembly assays, such as surface plasmonresonance (SPR) assays described in Forneris et. al. or Katschke et.al., or similar E-S assembly assays assessed by ELISA or similar assays.

The anti-fD aptamers as disclosed herein, in some cases, may bind to theexosite of fD so as to prevent formation of the ES complex. Withoutwishing to be bound by theory, the high specificity of fD for fB may bedue to protein-protein interactions between the exosites of fD and fB.The exosite of fD is approximately 25 Å from the catalytic center andconsists of 4 loops comprised by residues 145-149, 169-173, 185-188 and220-224. In some cases, the anti-fD compounds of the disclosure maytarget the exosite of fD and prevent the interaction of fD with fB.Anti-fD compounds of this nature may target one or more of the 4 loopsof the fD exosite, for example, the anti-fD compounds may be designed totarget one or more of amino acid residues 145-149, 169-173, 185-188 and220-224 of fD. In cases where an anti-fD aptamer binds to one or more ofamino acid residues 145-149, 169-173, 185-188, and 220-224, it may besaid that such aptamers bind to the exosite of fD.

Aptamer inhibitors that block binding of the C3bB substrate to fD mayinhibit alternative complement dependent hemolysis of red blood cells.Such inhibitors may enhance the esterase activity of fD againstthioester substrates of fD such as Z-Lys-S-Bzl, as observed for theanti-fD Fab's when bound to human fD (Katschke et. al.). Alternatively,aptamers which bind to the exosite of fD may not impact the esteraseactivity of fD, as for example, when the anti-fD Fab in Katschke et. al.binds fD from cynomolgus monkeys, it neither inhibits nor enhances fDesterase activity. Exosite binding aptamers would inhibit fB cleavage inthe C3bB complex by fD. In esterase assays, such inhibitors may increasek_(cat) and have no or minimal impact on K_(m) of fD, with the primaryeffect increasing k_(cat) and increasing k_(cat)/K_(m), or suchinhibitors would neither impact k_(cat) or K_(m) or k_(cat)/K_(m). Incomplete biochemical assays, such inhibitors would primarily increaseK_(m) and decrease k_(cat)/K_(m). Such inhibitors may prevent formationof the enzyme-substrate complex (fD-C3bB complex) as assessed inenzymatic assays or enzyme-substrate assembly assays, such as surfaceplasmon resonance (SPR) assays described in Forneris et. al. or Katschkeet. al., or similar ES assembly assays assessed by ELISA or similarassays.

Catalytic turn-over of fD activation of fB requires dissociation of theES complex if bound in a non-productive state or the EP (fD-C3bBb)complex upon fB cleavage. The anti-fD aptamers as disclosed herein, insome cases, may bind to fD in such a way as to prevent dissociation offD from C3bB or C3bBb. As envisioned, such aptamers may bind near theexosite of fD and bind to fD in such a manner as to increase theaffinity of fD for C3bB or C3bBb by decreasing the off-rate of thisinteraction. Such aptamers could be generated by selection against thefD-C3bB complex, by for example using a catalytically inactivated formof fD such as a mutant form in which Ser195 is mutated to Ala195(Forneris et. al.), to provide a stable, non-reactive ES complex as atarget for selection. Aptamers possessing such a mechanism of actionwould inhibit alternative complement dependent hemolysis of red bloodcells. Such inhibitors may inhibit the esterase activity of fD againstthioester substrates of fD such as Z-Lys-S-Bzl, or may not impact theesterase activity of fD. Such binding aptamers would inhibit theturn-over of fB cleavage in the C3bB complex by fD. In esterase assays,such inhibitors may decrease the k_(cat) and have no or minimal impacton K_(m) of fD, with the primary effect decreasing k_(cat) anddecreasing k_(cat)/K_(m), or such inhibitors would neither impactk_(cat) or K_(m) or k_(cat)/K_(m). In complete biochemical assays, suchinhibitors would primarily decrease K_(cat) and decrease k_(cat)/K_(m).Such inhibitors would enhance formation of the enzyme-substrate complex(fD-C3bB complex) as assessed in enzymatic assays or enzyme-substrateassembly assays, such as surface plasmon resonance (SPR) assaysdescribed in Forneris et. al., and may increase the apparent affinity offD for C3bB or C3bBb.

In some cases, an aptamer as described herein may bind the same epitopeas an anti-fD antibody or antibody fragment thereof. In some cases, anaptamer as described herein may bind to the same epitope as an anti-fDtherapeutic antibody. For example, the anti-fD aptamer may bind to thesame or similar region of fD to that which an anti-fD therapeuticantibody such as an anti-fD Fab with an amino acid sequence of heavychain variable region according to SEQ ID NO:7 and an amino acidsequence of light chain variable region according to SEQ ID NO:8; Mab166-3 or LS-C135735 bind. For example, an anti-fD Fab with an amino acidsequence of heavy chain variable region according to SEQ ID NO:7 andlight chain variable region according to SEQ ID NO:8 may bind residues129-132, residues 164-178, Arg223 and Lys224, with the bulk of theinteraction involving the loop encompassing amino acid 170 (the “170loop”). In some cases, an aptamer that binds to the same or similarregion of fD to that which an anti-fD Fab with an amino acid sequence ofheavy chain variable region according to SEQ ID NO:7 and light chainvariable region according to SEQ ID NO:8 binds (e.g., a regioncomprising one or more of amino acid residues 129-132, 164-178, Arg223and Lys224) may be said to be binding to the exosite of fD.

In some cases, an anti-fD aptamer for the modulation of fD is provided.In some cases, an anti-fD aptamer for the inhibition of a functionassociated with fD is provided. In some cases, the anti-fD aptamerinhibits the catalytic activity of fD. In some cases, an anti-fD aptamerfor the treatment of dry AMD or geographic atrophy is provided. In somecases, an anti-fD aptamer for the treatment of wet AMD is provided. Insome cases, an anti-fD aptamer for the treatment of Stargardt disease isprovided.

The dissociation constant (K_(d)) can be used to describe the affinityof an aptamer for a target (or to describe how tightly the aptamer bindsto the target) or to describe the affinity of an aptamer for a specificepitope of a target (e.g., exosite, catalytic cleft, etc.). Thedissociation constant is defined as the molar concentration at whichhalf of the binding sites of a target are occupied by the aptamer. Thus,the smaller the K_(d), the tighter the binding of the aptamer to itstarget. In some cases, an anti-fD aptamer has a dissociation constant(K_(d)) for fD protein of less than 1 mM, less than 100 μM, less than 10μM, less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM,less than 500 pM, or less than 100 pM. In some cases, an anti-fD aptamerhas a dissociation constant (K_(d)) for fD protein of less than 50 nM.In some cases, an anti-fD aptamer has a dissociation constant (K_(d))for fD protein of less than 25 nM. In some cases, an anti-fD aptamer hasa dissociation constant (K_(d)) for fD protein of less than 10 nM. Insome cases, an anti-fD aptamer has a dissociation constant (K_(d)) forfD protein of less than 5 nM. In some cases, an anti-fD aptamer has adissociation constant (K_(d)) for fD protein of less than 500 pM. Insome cases, an anti-fD aptamer has a dissociation constant (K_(d)) forfD protein of less than 50 pM. In some cases, an anti-fD aptamer has adissociation constant (K_(d)) for fD protein of less than 5 pM. In somecases, the aptamer binds to the catalytic cleft, the active site, theexosite, and/or the self-inhibitory loop of fD with a K_(d) of less thanabout 1 mM, 100 μM, 10 μM, 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 500 pM, 50pM, or 5 pM. In some cases, the anti-fD aptamer binds to the catalyticcleft, the active site, and/or the self-inhibitory loop of fD with aK_(d) from about 500 pM to about 1 nM. In some cases, the anti-fDaptamer binds to the catalytic cleft, the active site, and/or theself-inhibitory loop of fD with a K_(d) from about 1 nM to about 10 nM.In some cases, the K_(d) is determined by a flow cytometry assay asdescribed herein.

The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 50 nM and have an IC₅₀ of less than about 50nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 50 nM and have an IC₅₀ of less than about 10nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 50 nM and have an IC₅₀ of less than about 5nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 10 nM and have an IC₅₀ of less than about 50nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 10 nM and have an IC₅₀ of less than about 10nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 10 nM and have an IC₅₀ of less than about 5nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 5 nM and have an IC₅₀ of less than about 50nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 5 nM and have an IC₅₀ of less than about 10nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of less than about 5 nM and have an IC₅₀ of less than about 5 nMas measured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the catalytic cleft of fD with aK_(d) of less than about 1 nM and have an IC₅₀ of less than about 50 nMas measured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the catalytic cleft of fD with aK_(d) of less than about 1 nM and have an IC₅₀ of less than about 10 nMas measured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the catalytic cleft of fD with aK_(d) of less than about 1 nM and have an IC₅₀ of less than about 5 nMas measured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the catalytic cleft of fD with aK_(d) of about 500 pM or greater and have an IC₅₀ of less than about 50nM as measured by an alternative complement dependent hemolysis assay.The aptamers disclosed herein may bind to the catalytic cleft of fD witha K_(d) of about 500 pM or greater and have an IC₅₀ of less than about10 nM as measured by an alternative complement dependent hemolysisassay. The aptamers disclosed herein may bind to the catalytic cleft offD with a K_(d) of about 500 pM or greater and have an IC₅₀ of less thanabout 5 nM as measured by an alternative complement dependent hemolysisassay.

The aptamers disclosed herein may bind to the active site of fD with aK_(d) of less than about 50 nM and have an IC₅₀ of less than about 50 nMas measured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the active site of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay.

The aptamers disclosed herein may bind to a region of fD such that theaptamers block or occlude access to the active site of fD, therebypreventing a natural substrate of fD from accessing the active site. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay.

The aptamers disclosed herein may bind to the exosite of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 50 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 50 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 10 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 10 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 10 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 5 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 5 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 5 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 1 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 1 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofless than about 1 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofabout 500 pM or greater and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofabout 500 pM or greater and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the exosite of fD with a K_(d) ofabout 500 pM or greater and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay.

The aptamers disclosed herein may bind to a region of fD such that theaptamers block or occlude access to the substrate-binding exosite of fD.The aptamers disclosed herein may bind to such a region of fD with aK_(d) of less than about 50 nM and have an IC₅₀ of less than about 50 nMas measured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 50 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 5 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of less than about 1 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 10 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to such a region of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay.

The aptamers disclosed herein may bind to a region of fD such that theaptamers block or occlude access to both the active site and thesubstrate-binding exosite of fD. The aptamers disclosed herein may bindto such a region of fD with a K_(d) of less than about 50 nM and have anIC₅₀ of less than about 50 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 50 nM and have anIC₅₀ of less than about 10 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 50 nM and have anIC₅₀ of less than about 5 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 10 nM and have anIC₅₀ of less than about 50 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 10 nM and have anIC₅₀ of less than about 10 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 10 nM and have anIC₅₀ of less than about 5 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 5 nM and have anIC₅₀ of less than about 50 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 5 nM and have anIC₅₀ of less than about 10 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 5 nM and have anIC₅₀ of less than about 5 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 1 nM and have anIC₅₀ of less than about 50 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 1 nM and have anIC₅₀ of less than about 10 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of less than about 1 nM and have anIC₅₀ of less than about 5 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of about 500 pM or greater and have anIC₅₀ of less than about 50 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of about 500 pM or greater and have anIC₅₀ of less than about 10 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind tosuch a region of fD with a K_(d) of about 500 pM or greater and have anIC₅₀ of less than about 5 nM as measured by an alternative complementdependent hemolysis assay.

The aptamers disclosed herein may bind to the self-inhibitory loop of fDwith a K_(d) of less than about 50 nM and have an IC₅₀ of less thanabout 50 nM as measured by an alternative complement dependent hemolysisassay. The aptamers disclosed herein may bind to the self-inhibitoryloop of fD with a K_(d) of less than about 50 nM and have an IC₅₀ ofless than about 10 nM as measured by an alternative complement dependenthemolysis assay. The aptamers disclosed herein may bind to theself-inhibitory loop of fD with a K_(d) of less than about 50 nM andhave an IC₅₀ of less than about 5 nM as measured by an alternativecomplement dependent hemolysis assay. The aptamers disclosed herein maybind to the self-inhibitory loop of fD with a K_(d) of less than about10 nM and have an IC₅₀ of less than about 50 nM as measured by analternative complement dependent hemolysis assay. The aptamers disclosedherein may bind to the self-inhibitory loop of fD with a K_(d) of lessthan about 10 nM and have an IC₅₀ of less than about 10 nM as measuredby an alternative complement dependent hemolysis assay. The aptamersdisclosed herein may bind to the self-inhibitory loop of fD with a K_(d)of less than about 10 nM and have an IC₅₀ of less than about 5 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the self-inhibitory loop of fDwith a K_(d) of less than about 5 nM and have an IC₅₀ of less than about50 nM as measured by an alternative complement dependent hemolysisassay. The aptamers disclosed herein may bind to the self-inhibitoryloop of fD with a K_(d) of less than about 5 nM and have an IC₅₀ of lessthan about 10 nM as measured by an alternative complement dependenthemolysis assay. The aptamers disclosed herein may bind to theself-inhibitory loop of fD with a K_(d) of less than about 5 nM and havean IC₅₀ of less than about 5 nM as measured by an alternative complementdependent hemolysis assay. The aptamers disclosed herein may bind to theself-inhibitory loop of fD with a K_(d) of less than about 1 nM and havean IC₅₀ of less than about 50 nM as measured by an alternativecomplement dependent hemolysis assay. The aptamers disclosed herein maybind to the self-inhibitory loop of fD with a K_(d) of less than about 1nM and have an IC₅₀ of less than about 10 nM as measured by analternative complement dependent hemolysis assay. The aptamers disclosedherein may bind to the self-inhibitory loop of fD with a K_(d) of lessthan about 1 nM and have an IC₅₀ of less than about 5 nM as measured byan alternative complement dependent hemolysis assay. The aptamersdisclosed herein may bind to the self-inhibitory loop of fD with a K_(d)of about 500 pM or greater and have an IC₅₀ of less than about 50 nM asmeasured by an alternative complement dependent hemolysis assay. Theaptamers disclosed herein may bind to the self-inhibitory loop of fDwith a K_(d) of about 500 pM or greater and have an IC₅₀ of less thanabout 10 nM as measured by an alternative complement dependent hemolysisassay. The aptamers disclosed herein may bind to the self-inhibitoryloop of fD with a K_(d) of about 500 pM or greater and have an IC₅₀ ofless than about 5 nM as measured by an alternative complement dependenthemolysis assay.

In some aspects, the aptamers disclosed herein have an improvedhalf-life as compared to other therapeutics, including antibodies. Insome cases, the aptamers have an improved half-life in a biologicalfluid or solution as compared to an antibody. In some cases, theaptamers have an improved half-life in vivo as compared to an antibody.In one example, the aptamers have an improved half-life when injectedinto the eye (intraocular half-life) as compared to an antibody. In somecases, the aptamers may have an improved intraocular half-life wheninjected into the eye of a human. In some cases, the aptamers maydemonstrate improved stability over antibodies, under physiologicalconditions.

In some cases, the aptamers described herein have an intraocularhalf-life of at least 7 days in a human. In some cases, the aptamersdescribed herein have an intraocular half-life of at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, at least 14 days, at least 15 days, at least 20 days orgreater in a human.

In some cases, the aptamers described herein have an intraocularhalf-life of at least 1 day in a non-human animal (e.g.,rodent/rabbit/monkey). In some cases, the aptamers described herein havean intraocular half-life of at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, at least 7days, at least 8 days, at least 9 days, at least 10 days or greater in anon-human animal such as a rodent, rabbit or monkey.

In some aspects, the aptamers described herein may have a shorterhalf-life as compared to other therapeutics. For example, an unmodifiedor unconjugated aptamer may have a lower half-life as compared to amodified or conjugated aptamer, however, the low molecular weight of theunmodified or unconjugated forms may allow for orders of magnitudegreater initial concentrations, thereby achieving greaterduration/efficacy. In some examples, the aptamer may have an intraocularhalf-life of less than about 7 days in a human. In some examples, theaptamers described herein have an intraocular half-life of less thanabout 6 days, less than about 5 days or even less than about 4 days in ahuman.

The aptamers disclosed herein may demonstrate high specificity for fDversus other complement pathway components. Generally, the aptamer maybe selected such that the aptamer has high affinity for fD, but withlittle to no affinity for other complement pathway components or serineproteases. In some cases, the aptamers bind to fD with a specificity ofat least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold,or greater than 20-fold greater than the aptamers bind to any of C3, CS,Factor B, Factor H or Factor I (or any of their related dimeric,trimeric, or multimeric complexes, units or subunits) at relative serumconcentrations. For example, in some cases the aptamers bind to fD witha specificity of at least 50-fold greater than the aptamers bind to anyof C3, C5, Factor B, Factor H or Factor I (or any of their relateddimeric, trimeric, or multimeric complexes, units or subunits) atrelative serum concentrations. For example, in some cases the aptamersbind to FD with a specificity of at least 100-fold greater than theaptamers bind to any of C3, C5, Factor B, Factor H or Factor I (or anyof their related dimeric, trimeric, or multimeric complexes, units orsubunits) at relative serum concentrations.

The activity of a therapeutic agent can be characterized by the halfmaximal inhibitory concentration (IC₅₀). The IC₅₀ is calculated as theconcentration of therapeutic agent in nM at which half of the maximuminhibitory effect of the therapeutic agent is achieved. The IC₅₀ isdependent upon the assay utilized to calculate the value. In someexamples, the IC₅₀ of an aptamer described herein is less than 100 nM,less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, lessthan 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM asmeasured by an alternative complement dependent hemolysis assay(Pangburn, 1988, Methods in Enzymology; and Katschke, 2009, Journal ofBiological Chemistry).

In some examples, the aptamers described herein increase the activity offD as measured by a fD esterase activity assay as compared to a control,and inhibit activity of fD as measured by an alternative complementdependent hemolysis assay. In other examples, the aptamers describedherein inhibit activity of fD as measured by a fD esterase activityassay as compared to a control, and inhibit activity of fD as measuredby an alternative complement dependent hemolysis assay. In yet othercases, the aptamer does not inhibit activity of fD as measured by a fDesterase activity assay as compared to a control, and does inhibitactivity of fD as measured by an alternative complement dependenthemolysis assay.

Aptamers generally have high stability at ambient temperatures forextended periods of time. The aptamers described herein demonstrategreater than 70%, greater than 75%, greater than 80%, greater than 85%,greater 90%, greater than 91%, greater than 92%, greater than 93%,greater than 94%, greater than 95%, greater than 96%, greater than 97%,greater than 98%, greater than 99%, greater than 99.5%, or greater than99.9% activity in solution under physiological conditions at 30 days orlater.

In some cases, a composition of the disclosure comprises anti-fDaptamers, wherein essentially 100% of the anti-fD aptamers comprisenucleotides having ribose in the β-D-ribofuranose configuration. Inother examples, a composition of the disclosure may comprise anti-fDaptamers, wherein at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or greater than 90% of the anti-fD aptamers haveribose in the β-D-ribofuranose configuration.

Indications

In some aspects, the methods and compositions provided herein are usedfor the treatment of ocular diseases or disorders. Ocular diseases ordisorders can include, without limitation, any complement-mediatedocular disorders such as inflammatory conjunctivitis, including allergicand giant papillary conjunctivitis, macular edema, uveitis,endophthalmitis, scleritis, corneal ulcers, dry eye syndrome, glaucoma,ischemic retinal disease, corneal transplant rejection, complicationsrelated to intraocular surgery such intraocular lens implantation andinflammation associated with cataract surgery, Behcet's disease,Stargardt disease, immune complex vasculitis, Fuch's disease,Vogt-Koyanagi-Harada disease, subretinal fibrosis, keratitis,vitreo-retinal inflammation, ocular parasitic infestation/migration,retinitis pigmentosa, cytomeglavirus retinitis and choroidalinflammation.

Other examples of ocular diseases or disorders that may be amendable totreatment by the methods and compositions provided herein may include,without limitation, ectropion, lagophthalmos, blepharochalasis, ptosis,xanthelasma of the eyelid, parasitic infestation of the eyelid,dermatitis of the eyelid, dacryoadenitis, epiphora, dysthyroidexophthalmos, conjunctivitis, scleritis, keratitis, corneal ulcer,corneal abrasion, snow blindness, arc eye, Thygeson's superficialpunctate keratopathy, corneal neovascularization, Fuchs' dystrophy,keratoconus, keratoconjunctivitis sicca, iritis, uveitis, sympatheticophthalmia, cataracts, chorioretinal inflammation, focal chorioretinalinflammation, focal chorioretinitis, focal choroiditis, focal retinitis,focal retinochoroiditis, disseminated chorioretinal inflammation,disseminated chorioretinitis, disseminated choroiditis, disseminatedretinitis, disseminated retinochoroiditis, exudative retinopathy,posterior cyclitis, pars planitis, Harada's disease, chorioretinalscars, macula scars of posterior pole, solar retinopathy, choroidaldegeneration, choroidal atrophy, choroidal sclerosis, angioid streaks,hereditary choroidal dystrophy, choroideremia, choroidal dystrophy(central arealor), gyrate atrophy (choroid), ornithinaemia, choroidalhaemorrhage and rupture, choroidal haemorrhage (not otherwisespecified), choroidal haemorrhage (expulsive), choroidal detachment,retinoschisis, retinal artery occlusion, retinal vein occlusion,hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathyof prematurity, macular degeneration, Bull's Eye maculopathy, epiretinalmembrane, peripheral retinal degeneration, hereditary retinal dystrophy,retinitis pigmentosa, retinal haemorrhage, separation of retinal layers,central serous retinopathy, retinal detachment, macular edema,glaucoma—optic neuropathy, glaucoma suspect—ocular hypertension, primaryopen-angle glaucoma, primary angle-closure glaucoma, floaters, Leber'shereditary optic neuropathy, optic disc drusen, strabismus,ophthalmoparesis, progressive external ophthaloplegia, esotropia,exotropia, disorders of refraction and accommodation, hypermetropia,myopia, astigmastism, anisometropia, presbyopia, internalophthalmoplegia, amblyopia, Leber's congenital amaurosis, scotoma,anopsia, color blindness, achromatopsia, maskun, nyctalopia, blindness,River blindness, micropthalmia, coloboma, red eye, Argyll Robertsonpupil, keratomycosis, xerophthalmia, aniridia, sickle cell retinopathy,ocular neovascularization, retinal neovascularization, subretinalneovascularization; rubeosis iritis inflammatory diseases, chronicposterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma,neovascular glaucoma; neovascularization resulting following a combinedvitrectomy-2 and lensectomy, vascular diseases, retinal ischemia,choroidal vascular insufficiency, choroidal thrombosis,neovascularization of the optic nerve, diabetic macular edema, cystoidmacular edema, proliferative vitreoretinopathy, and neovascularizationdue to penetration of the eye or ocular injury.

In some aspects, the methods and compositions provided herein aresuitable for the treatment of macular degeneration. In some cases,macular degeneration is age-related macular degeneration. In some cases,the methods and compositions can be utilized to treat neovascular orexudative (“wet”) age-related macular degeneration. In other cases, themethods and compositions can be utilized to treat non-exudative (“dry”)age-related macular degeneration. In some cases, advanced forms of dryage-related macular degeneration can be treated, including geographicatrophy. In some cases, the methods and compositions herein can beutilized to prevent age-related macular degeneration and associateddiseases thereof. In other cases, the methods and compositions hereincan be utilized to slow or halt the progression of age-related maculardegeneration and associated diseases thereof.

In some aspects, the methods and compositions provided herein aresuitable for the treatment of Stargardt disease. In some cases, themethods and compositions herein can be utilized to prevent age-relatedStargardt disease. In other cases, the methods and compositions hereincan be utilized to slow or halt the progression of Stargardt disease.

In some aspects, the methods and compositions provided herein aresuitable for the treatment of diseases causing ocular symptoms. Examplesof symptoms which may be amenable to treatment with the methodsdisclosed herein include: increased drusen volume, reduced readingspeed, reduced color vision, retinal thickening, increase in centralretinal volume and/or, macular sensitivity, loss of retinal cells,increase in area of retinal atrophy, reduced best corrected visualacuity such as measured by Snellen or ETDRS scales, Best CorrectedVisual Acuity under low luminance conditions, impaired night vision,impaired light sensitivity, impaired dark adaptation, contrastsensitivity, and patient reported outcomes.

In some cases, the methods and compositions provided herein mayalleviate or reduce a symptom of a disease. In some cases, treatmentwith an aptamer provided herein may result in a reduction in theseverity of any of the symptoms described herein. In some cases,treatment with an aptamer described herein may slow, halt or reverse theprogression of any of the symptoms described herein. In some cases,treatment with an aptamer described herein may prevent the developmentof any of the symptoms described herein. In some cases, treatment withan aptamer described herein may slow, halt or reverse the progression ofa disease, as measured by the number and severity of symptomsexperienced. Examples of symptoms and relevant endpoints where theaptamer may have a therapeutic effect include increased drusen volume,reduced reading speed, reduced color vision, retinal thickening,increase in central retinal volume and/or, macular sensitivity, loss ofretinal cells, increase in area of retinal atrophy, reduced bestcorrected visual acuity such as measured by Snellen or ETDRS scales,Best Corrected Visual Acuity under low luminance conditions, impairednight vision, impaired light sensitivity, impaired dark adaptation,contrast sensitivity, and patient reported outcomes. In some instances,treatment with an aptamer described herein may have beneficial effectsas measured by clinical endpoints including drusen volume, readingspeed, retinal thickness as measured by Optical Coherence Tomography orother techniques, central retinal volume, number and density of retinalcells, area of retinal atrophy as measured by Fundus Photography orFundus Autofluoresence or other techniques, best corrected visual acuitysuch as measured by Snellen or ETDRS scales, Best Corrected VisualAcuity under low luminance conditions, light sensitivity, darkadaptation, contrast sensitivity, and patient reported outcomes asmeasured by such tools as the National Eye Institute Visual FunctionQuestionnaire and Health Related Quality of Life Questionnaires.

Subjects

The terms “subject” and “patient” are used interchangeably herein torefer to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans,research animals, farm animals, sport animals, and pets. In some cases,the methods described herein may be used on tissues or cells derivedfrom a subject and the progeny of such tissues or cells. For example,aptamers described herein may be used to effect some function (e.g.,inhibit or block fD) in tissues or cells of a subject. The tissues orcells may be obtained from a subject in vivo. In some cases, the tissuesor cells are cultured in vitro and contacted with a composition providedherein (e.g., an aptamer).

In some aspects, the methods and compositions provided herein are usedto treat a subject in need thereof. In some cases, the subject suffersfrom an ocular disease or disorder. In some cases, the subject is ahuman. In some cases, the human is a patient at a hospital or a clinic.In some cases, the subject is a non-human animal, for example, anon-human primate, a livestock animal, a domestic pet, or a laboratoryanimal. For example, a non-human animal can be an ape (e.g., achimpanzee, a baboon, a gorilla, or an orangutan), an old world monkey(e.g., a rhesus monkey), a new world monkey, a dog, a cat, a bison, acamel, a cow, a deer, a pig, a donkey, a horse, a mule, a lama, a sheep,a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or anyother non-human animal.

In cases where the subject is a human, the subject may be of any age. Insome cases, the subject has an age-related ocular disease or disorder(e.g., age-related macular degeneration, Stargardt disease). In somecases, the subject is about 50 years or older. In some cases, thesubject is about 55 years or older. In some cases, the subject is about60 years or older. In some cases, the subject is about 65 years orolder. In some cases, the subject is about 70 years or older. In somecases, the subject is about 75 years or older. In some cases, thesubject is about 80 years or older. In some cases, the subject is about85 years or older. In some cases, the subject is about 90 years orolder. In some cases, the subject is about 95 years or older. In somecases, the subject is about 100 years or older. In some cases, thesubject is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100 or greater than 100 years old. In some cases, the subject is about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 orgreater than 20 years old.

In cases where the subject is a human, the subject may have any geneticprofile. In some cases, the subject may have mutations in complementFactor H (CFH), complement component 3 (C3), complement component 2(C2), complement Factor B, complement Factor I (CFI), ABC4A, ELOVL4, orany combination thereof.

In some aspects, the methods and compositions provided herein areutilized to treat a subject suffering from ocular symptoms as describedherein. In some aspects, the methods and compositions provided hereinare utilized to treat a subject suffering from an ocular disease asprovided herein. In some cases, the methods and compositions providedherein are utilized to treat a subject suffering from wet AMD. In somecases, the methods and compositions provided herein are utilized totreat a subject suffering from dry AMD or geographic atrophy. In somecases, the methods and compositions provided herein are utilized totreat a subject suffering from Stargardt disease.

In some aspects, the methods and compositions provided herein may beutilized to treat a subject with a highly active immune system. In somecases, the methods and compositions provided herein may be used to treata subject with an autoimmune disease. In some cases, the methods andcompositions provided herein may be used to treat a subject with aninflammatory disease. In some cases, the methods and compositionsprovided herein may be used to treat a subject undergoing aninflammatory reaction to a disease such as an infectious disease. Forexample, the aptamers described herein may be used to treat a subjectwith a fever. In some cases, the aptamers described herein may be usedto treat a subject with an allergy. In some cases, the aptamersdescribed herein may be used to treat a subject suffering from anallergic response. In some cases, the aptamers described herein may beparticularly useful for treating a subject who has experienced anallergic reaction to an antibody treatment, and/or who has developedneutralizing antibodies against an antibody treatment.

Pharmaceutical Compositions or Medicaments

Disclosed herein are pharmaceutical compositions or medicaments, usedinterchangeably, for use in a method of therapy, or for use in a methodof medical treatment. Such use may be for the treatment of oculardiseases. In some cases, the pharmaceutical compositions can be used totreat AMD. In some cases, the pharmaceutical compositions can be used totreat non-exudative (dry) AMD. In some cases, the pharmaceuticalcompositions can be used to treat geographic atrophy (advanced dry AMD).In some cases, the pharmaceutical compositions can be used to treat wetAMD. In some cases, the pharmaceutical compositions can be used to treatStargardt disease. Pharmaceutical compositions described herein mayinclude one or more aptamers for the treatment of dry AMD.Pharmaceutical compositions described herein may include one or moreaptamers for the treatment of wet AMD. Pharmaceutical compositionsdescribed herein may include one or more aptamers for the treatment ofStargardt disease. In some cases, the one or more aptamers bind to andinhibit a component of the alternative complement pathway. In somecases, the one or more aptamers bind to one or more targets of fD asdescribed herein. In some cases, the one or more aptamers inhibit fD asdescribed herein. In some cases, the compositions include, e.g., aneffective amount of the aptamer, alone or in combination, with one ormore vehicles (e.g., pharmaceutically acceptable compositions or e.g.,pharmaceutically acceptable carriers). In some cases, the compositionsdescribed herein are administered with one or more additionalpharmaceutical treatments (e.g., co-administered, sequentiallyadministered or co-formulated). In some examples, the compositionsdescribed herein are co-administered with one or more of ananti-vascular endothelial growth factor (VEGF) therapy, an anti-Factor Ptherapy, an anti-complement component 5 (C5) therapy, an anti-complementcomponent 3 (C3) therapy, an anti-platelet-derived growth factor (PDGF)therapy, an anti-hypoxia-inducible factor 1-alpha (HIF1a) therapy, ananti-FAS therapy, an anti-integrin therapy or an anti-angiopoietin-2(Ang2) therapy.

Formulations

Compositions as described herein may comprise a liquid formulation, asolid formulation or a combination thereof. Non-limiting examples offormulations may include a tablet, a capsule, a gel, a paste, a liquidsolution and a cream. The compositions of the present disclosure mayfurther comprise any number of excipients. Excipients may include anyand all solvents, coatings, flavorings, colorings, lubricants,disintegrants, preservatives, sweeteners, binders, diluents, andvehicles (or carriers). Generally, the excipient is compatible with thetherapeutic compositions of the present disclosure. The pharmaceuticalcomposition may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and other substances such as, for example, sodium acetate, andtriethanolamine oleate.

Dosage and Routes of Administration

Therapeutic doses of formulations of the disclosure can be administeredto a subject in need thereof. In some cases, a formulation isadministered to the eye of a subject to treat, for example, dry AMD,geographic atrophy, wet AMD or Stargardt disease. Administration to theeye can be a) topical; b) local ocular delivery; or c) systemic. Atopical formulation can be applied directly to the eye (e.g., eye drops,contact lens loaded with the formulation) or to the eyelid (e.g., cream,lotion, gel). In some cases, topical administration can be to a siteremote from the eye, for example, to the skin of an extremity. This formof administration may be suitable for targets that are not produceddirectly by the eye. In one non-limiting example, fD is thought to beproduced primarily by adipose cells, and thus an anti-fD aptamer may beadministered topically to a non-ocular region of the body. In somecases, a formulation of the disclosure is administered by local oculardelivery. Non-limiting examples of local ocular delivery includeintravitreal (IVT), intracamarel, subconjunctival, subtenon,retrobulbar, posterior juxtascleral, and peribulbar. In some cases, aformulation of the disclosure is delivered by intravitrealadministration (IVT). Local ocular delivery may generally involveinjection of a liquid formulation. In other cases, a formulation of thedisclosure is administered systemically. Systemic administration caninvolve oral administration. In some cases, systemic administration canbe intravenous administration, subcutaneous administration, infusion,implantation, and the like.

Other formulations suitable for delivery of the pharmaceuticalcompositions described herein may include a sustained release gel orpolymer formulations by surgical implantation of a biodegradablemicrosize polymer system, e.g., microdevice, microparticle, or sponge,or other slow release transscleral devices, implanted during thetreatment of an ophthalmic disease, or by an ocular delivery device,e.g. polymer contact lens sustained delivery device. In some cases, theformulation is a polymer gel, a self assembling gel, a durable implant,an eluting implant, a biodegradable matrix or biodegradable polymers. Insome cases, the formulation may be administered by iontophoresis usingelectric current to drive the composition from the surface to theposterior of the eye. In some cases, the formulation may be administeredby a surgically implanted port with an intravitreal reservoir, anextra-vitreal reservoir or a combination thereof. Examples ofimplantable ocular devices can include, without limitation, theDurasert™ technology developed by Bausch & Lomb, the ODTx devicedeveloped by On Demand Therapeutics, the Port Delivery System developedby ForSight VISION4 and the Replenish MicroPump™ System developed byReplenish, Inc.

In some cases, nanotechnologies can be used to deliver thepharmaceutical compositions including nanospheres, nanoparticles,nanocapsules, liposomes, nanomicelles and dendrimers.

A composition of the disclosure can be administered once or more thanonce each day. In some cases, the composition is administered as asingle dose (i.e., one-time use). In this example, the single dose maybe curative. In other cases, the composition may be administeredserially (e.g., taken every day without a break for the duration of thetreatment regimen). In some cases, the treatment regime can be less thana week, a week, two weeks, three weeks, a month, or greater than amonth. In some cases, the composition is administered over a period ofat least 12 weeks. In other cases, the composition is administered for aday, at least two consecutive days, at least three consecutive days, atleast four consecutive days, at least five consecutive days, at leastsix consecutive days, at least seven consecutive days, at least eightconsecutive days, at least nine consecutive days, at least tenconsecutive days, or at least greater than ten consecutive days. In somecases, a therapeutically effective amount can be administered one timeper week, two times per week, three times per week, four times per week,five times per week, six times per week, seven times per week, eighttimes per week, nine times per week, 10 times per week, 11 times perweek, 12 times per week, 13 times per week, 14 times per week, 15 timesper week, 16 times per week, 17 times per week, 18 times per week, 19times per week, 20 times per week, 25 times per week, 30 times per week,35 times per week, 40 times per, week, or greater than 40 times perweek. In some cases, a therapeutically effective amount can beadministered one time per day, two times per day, three times per day,four times per day, five times per day, six times per day, seven timesper day, eight times per day, nine times per day, 10 times per day, orgreater than 10 times per day. In some cases, the composition isadministered at least twice a day. In further cases, the composition isadministered at least every hour, at least every two hours, at leastevery three hours, at least every four hours, at least every five hours,at least every six hours, at least every seven hours, at least everyeight hours, at least every nine hours, at least every 10 hours, atleast every 11 hours, at least every 12 hours, at least every 13 hours,at least every 14 hours, at least every 15 hours, at least every 16hours, at least every 17 hours, at least every 18 hours, at least every19 hours, at least every 20 hours, at least every 21 hours, at leastevery 22 hours, at least every 23 hours, or at least every day.

Aptamers as described herein may be particularly advantageous overantibodies as they may sustain therapeutic intravitreal concentrationsof drug for longer periods of time, thus requiring less frequentadministration. For example, an anti-fD Fab having an amino acidsequence of heavy chain variable region according to SEQ ID NO:7 and alight chain variable region according to SEQ ID NO:8, may show clinicalefficacy for the treatment of geographic atrophy at 10 mg when dosedevery 4 weeks (q4w) but not every 8 weeks (q8w). The aptamers describedherein have a longer intraocular half-life, and/or sustain therapeuticintravitreal concentrations of drug for longer periods of time, than ananti-fD Fab with an amino acid sequence of heavy chain variable regionaccording to SEQ ID NO:7 and light chain variable region according toSEQ ID NO:8 and other antibody therapies and thus, can be dosed lessfrequently. In some cases, the aptamers are dosed at least every 4 weeks(q4w), every 5 weeks (q5w), every 6 weeks (q6w), every 7 weeks (q7w),every 8 weeks (q8w), every 9 weeks (q9w), every 10 weeks (g10w), every12 weeks (q12w) or greater than q12w.

In some aspects, a therapeutically effective amount of the aptamer isadministered. A “therapeutically effective amount” or “therapeuticallyeffective dose” are used interchangeably herein and refer to an amountof a therapeutic agent (e.g., an aptamer) that provokes a therapeutic ordesired response in a subject. The therapeutically effective amount ofthe composition may be dependent on the route of administration. In thecase of systemic administration, a therapeutically effective amount maybe about 10 mg/kg to about 100 mg/kg. In some cases, a therapeuticallyeffective amount may be about 10 μg/kg to about 1000 μg/kg for systemicadministration. For intravitreal administration, a therapeuticallyeffective amount can be about 0.01 mg to about 150 mg in about 25 μl toabout 100 μl volume per eye.

Methods

Disclosed herein are methods for the treatment of ocular diseases. Insome cases, the ocular disease is dry age-related macular degenerationor geographic atrophy. In some cases, the method involves administeringa therapeutically effective amount of a composition to a subject totreat the disease. In some cases, the composition includes one or moreaptamers as described herein. The aptamers may inhibit a functionassociated with fD as described herein. The methods can be performed ata hospital or a clinic, for example, the pharmaceutical compositions canbe administered by a health-care professional. In other cases, thepharmaceutical compositions can be self-administered by the subject.Treatment may commence with the diagnosis of a subject with an oculardisease (e.g., AMD). In the event that further treatments are necessary,follow-up appointments may be scheduled for the administration ofsubsequence doses of the composition, for example, administration every8 weeks.

Methods of Generating Aptamers The SELEX™ Method

The aptamers described herein can be generated by any method suitablefor generating aptamers. In some cases, the aptamers described hereinare generated by a process known as Systematic Evolution of Ligands byExponential Enrichment” (“SELEX™”). The SELEX™ process is described in,e.g., U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990,now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”,and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “NucleicAcid Ligands”, each of which are herein incorporated by reference. Byperforming iterative cycles of selection and amplification, SELEX™ maybe used to obtain aptamers with any desired level of target bindingaffinity.

The SELEX™ method relies as a starting point upon a large library orpool of single stranded oligonucleotides comprising randomizedsequences. The oligonucleotides can be modified or unmodified DNA, RNA,or DNA/RNA hybrids. In some examples, the pool comprises 100% random orpartially random oligonucleotides. In other examples, the pool comprisesrandom or partially random oligonucleotides containing at least onefixed sequence and/or conserved sequence incorporated within randomizedsequence. In other examples, the pool comprises random or partiallyrandom oligonucleotides containing at least one fixed sequence and/orconserved sequence at its 5′ and/or 3′ end which may comprise a sequenceshared by all the molecules of the oligonucleotide pool. Fixed sequencesare sequences common to oligonucleotides in the pool which areincorporated for a preselected purpose such as, CpG motifs,hybridization sites for PCR primers, promoter sequences for RNApolymerases (e.g., T3, T4, T7, and SP6), sequences to form stems topresent the randomized region of the library within a defined terminalstem structure, restriction sites, or homopolymeric sequences, such aspoly A or poly T tracts, catalytic cores, sites for selective binding toaffinity columns, and other sequences to facilitate cloning and/orsequencing of an oligonucleotide of interest. Conserved sequences aresequences, other than the previously described fixed sequences, sharedby a number of aptamers that bind to the same target.

The oligonucleotides of the pool can include a randomized sequenceportion as well as fixed sequences necessary for efficientamplification. Typically the oligonucleotides of the starting poolcontain fixed 5′ and 3′ terminal sequences which flank an internalregion of 30-50 random nucleotides. The randomized nucleotides can beproduced in a number of ways including chemical synthesis and sizeselection from randomly cleaved cellular nucleic acids. Sequencevariation in test nucleic acids can also be introduced or increased bymutagenesis before or during the selection/amplification iterations.

The random sequence portion of the oligonucleotide can be of any lengthand can comprise ribonucleotides and/or deoxyribonucleotides and caninclude modified or non-natural nucleotides or nucleotide analogs.Typical syntheses carried out on automated DNA synthesis equipment yield10¹⁴-10¹⁶ individual molecules, a number sufficient for mostSELEX™experiments. Sufficiently large regions of random sequence in thesequence design increases the likelihood that each synthesized moleculeis likely to represent a unique sequence.

The starting library of oligonucleotides may be generated by automatedchemical synthesis on a DNA synthesizer. To synthesize randomizedsequences, mixtures of all four nucleotides are added at each nucleotideaddition step during the synthesis process, allowing for randomincorporation of nucleotides. As stated above, in some cases, randomoligonucleotides comprise entirely random sequences; however, in othercases, random oligonucleotides can comprise stretches of nonrandom orpartially random sequences. Partially random sequences can be created byadding the four nucleotides in different molar ratios at each additionstep.

The starting library of oligonucleotides may be RNA, DNA, substitutedRNA or DNA or combinations thereof. In those instances where an RNAlibrary is to be used as the starting library it is typically generatedby synthesizing a DNA library, optionally PCR amplifying, thentranscribing the DNA library in vitro using T7 RNA polymerase ormodified T7 RNA polymerases, and purifying the transcribed library. Thenucleic acid library is then mixed with the target under conditionsfavorable for binding and subjected to step-wise iterations of binding,partitioning and amplification, using the same general selection scheme,to achieve virtually any desired criterion of binding affinity andselectivity. More specifically, starting with a mixture containing thestarting pool of nucleic acids, the SELEX™ method includes steps of: (a)contacting the mixture with the target under conditions favorable forbinding; (b) partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules; (c) dissociating thenucleic acid-target complexes; (d) amplifying the nucleic acidsdissociated from the nucleic acid-target complexes to yield aligand-enriched mixture of nucleic acids; and (e) reiterating the stepsof binding, partitioning, dissociating and amplifying through as manycycles as desired to yield highly specific, high affinity nucleic acidligands to the target molecule. In those instances where RNA aptamersare being selected, the SELEX′ method further comprises the steps of:(i) reverse transcribing the nucleic acids dissociated from the nucleicacid-target complexes before amplification in step (d); and (ii)transcribing the amplified nucleic acids from step (d) before restartingthe process.

Within a nucleic acid mixture containing a large number of possiblesequences and structures, there is a wide range of binding affinitiesfor a given target. Those which have the higher affinity (lowerdissociation constants) for the target are most likely to bind to thetarget. After partitioning, dissociation and amplification, a secondnucleic acid mixture is generated, enriched for the higher bindingaffinity candidates. Additional rounds of selection progressively favorthe best ligands until the resulting nucleic acid mixture ispredominantly composed of only one or a few sequences. These can then becloned, sequenced and individually tested as ligands or aptamers for 1)target binding affinity; and 2) ability to effect target function.

Cycles of selection and amplification are repeated until a desired goalis achieved. In the most general case, selection/amplification iscontinued until no significant improvement in binding strength isachieved on repetition of the cycle. The method is typically used tosample approximately 10¹⁴ different nucleic acid species but may be usedto sample as many as about 10¹⁸ different nucleic acid species.Generally, nucleic acid aptamer molecules are selected in a 5 to 20cycle procedure.

In some cases, the aptamers of the disclosure are generated using theSELEX™ method as described above. In other cases, the aptamers of thedisclosure are generated using any modification or variant of the SELEX™method.

In some cases, the aptamers described herein have been generated usingmethodologies to select for specific sites related to activity orfunction of a target protein. In some cases, the aptamers describedherein may be selected using methods that improve the chances ofselecting an aptamer with a desired function or desired binding site. Insome cases, the aptamers described herein are generated using methodsthat increase the chances of selecting an aptamer that binds to a regionof fD that serves as an epitope for an anti-fD therapeutic antibody,which anti-fD therapeutic antibody inhibits a function associated withfD.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1. Identification of Modified RNA Aptamers to fD A. Selection ofAnti-Factor D Aptamers

Anti-factor D (fD) aptamers were identified using an N30 library (N30S)comprised of a 30-nucleotide random region flanked by constant regionscontaining a built-in stem region as depicted in FIG. 7A. The sequencein italics represents the forward and reverse primer binding sites. Thebuilt-in stem region is underlined. FIG. 7B depicts a representation ofthe N30S library with the reverse oligo hybridized. For nucleasestability, the library was composed of 2′-fluoro-G (2′F GTP) and2′-O-methyl (2′OMe) A/C/U. FIG. 7C depicts structures of modifiednucleotides used to generate the N30S library for selection againsttarget fD. For simplicity, the nucleosides, and not the nucleotidetriphosphates are shown.

The library sequence (underlined sequences represent the built-in stem)and the sequence of oligos used to amplify the library are described inTable 4.

TABLE 4 Library sequence and sequence of oligos used to amplify thelibrary SEQ ID NO. Sequence (5′ to 3′) SEQ ID NO: 4 Library sequenceGGGAGTGTGTACGAGGCATTAGGCCGCC- (Total library length: N30- 89 bases)GGCGGCTTTGATACTTGATCGCCCTAGAAGC SEQ ID NO: 5 N30S.FTCTTAATACGACTCACTATAGGGAGTGTGTA CGAGGCATTA SEQ ID NO: 6 N30S.RGCTTCTAGGGCGATCAAGTATCA

The starting library was transcribed from a pool of ˜10¹⁴double-stranded DNA (dsDNA) molecules. The dsDNA library was generatedby primer extension using Klenow exo (−) DNA polymerase, the poolforward primer (N30S.F) and synthetic single-stranded DNA (ssDNA)molecule encoding the library. The dsDNA was subsequently converted to100% backbone modified RNA via transcription using a mixture of 2′F GTP,2′OMe ATP/CTP/UTP and a variant of T7 RNA polymerase in buffer optimizedto facilitate efficient transcription. Following transcription, RNAswere treated with DNAse to remove the template dsDNA and purified.

The selection targeting fD was facilitated by the use of a His-taggedrecombinant human complement Factor D protein and magnetic His capturebeads. Briefly, beads (the amount varied with the amount of targetprotein coupled) were washed three times with immobilization buffer (50mM sodium phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20) and wereresuspended in 50 μL of immobilization buffer. His-tagged recombinantfD, in immobilization buffer, was then added to the beads and incubatedat room temperature for 30 minutes. The amount of target protein variedwith the rounds (Table 5). The beads were washed three times withbinding buffer SB1T (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 1 mMMgCl₂, 1 mM CaCl₂, 0.05% Tween-20) to remove any unbound protein andthen re-suspended in 50 μL SB1T buffer containing 1 μg/μ1 ssDNA and 0.1%BSA.

For the first round of selection, ˜3 nanomoles of the Round 0 RNA pool,˜10¹⁴ sequences, was used. Prior to each round, the library wasthermally equilibrated by heating at 80° C. for 5 minutes and cooled atroom temperature for 15 minutes in the presence of a 1.5-fold molarexcess of reverse primer (N30S.R) to allow the library to refold andsimultaneously block the 3′ end of the pool. Following renaturation, thefinal volume of the reaction was adjusted to 50 μL in SB1T supplementedwith 1 μg/ml ssDNA and 0.1% BSA.

For the first round, the library was added to the fD immobilized onbeads and incubated at 37° C. for 1 hour with intermittent mixing. Afterone hour, the beads were washed using 3×1 ml SB1T buffer to removeunbound aptamers. For round 0, each wash step was incubated for 5minutes. After washing, fD-bound aptamers were eluted using 200 μLelution buffer (2M Guanidine-HCl in SB buffer) two times (total volume400 μL). The eluted aptamers, in 400 μL of elution buffer, wereprecipitated by adding 40 μL 3M NaOAc, pH 5.2, 1 ml ethanol and 2 μlglycogen and incubating at −80° C. for 15 minutes. The recovered librarywas converted to DNA by reverse transcription using Super Script IVreverse transcriptase, and the ssDNA was subsequently amplified by PCR.The resulting dsDNA library was subsequently converted back intomodified RNA via transcription as described above. DNased, purified RNAwas used for subsequent rounds.

For subsequent rounds, the washing time and number of washes was variedas the selection progressed, the input RNA was kept fixed at 25picomoles, and the protein input varied (Table 5). After the firstround, a negative selection step was included in all the subsequentrounds. For the negative selection, the pool was prepared as describedbefore and first incubated with non-labelled beads for 1 hour at 37° C.in SB1T buffer. The beads were then spun down and the supernatantcontaining molecules that did not bind to the unlabeled beads wasincubated with fD-labeled beads for an additional 1 hour at 37° C.

B. Assessing the Progress of Selection

Flow cytometry was used to assess the progress of the selection. Forthese assays, RNA from each round was first hybridized with reversecomplement oligonucleotide composed of 2′OMe RNA labeled with Dylight®650 (Dy650-N30S.R.OMe). Briefly, the library was combined with 1.5-foldmolar excess of Dy650-N30S.R.OMe, heated at 80° C. for 6 minutes andallowed to cool at room temperature for 15 min. after which it wasincubated with beads labelled with fD, in SB1T buffer containing 0.1%BSA and 1 ssDNA. Following incubation for 1 hour at 37° C., the beadswere washed 3 times with SB1T, re-suspended in SB1T buffer and analyzedby flow cytometry. As shown in FIG. 8, an improvement in fluorescentsignal with the progressing rounds was seen as early as Round 3. AfterRound 6, there was little change in the binding signal through Round 8.“Beads” refers to the signal of fD-labelled beads in the absence oflabeled RNA. The apparent affinity of rounds 6, 7, and 8 for fD was alsomeasured using flow cytometry-based assays and revealed K_(d)s in therange of 8-45 nM (FIG. 9, Table 7).

C. Selection, Purification and Characterization of Clones

The enriched aptamer populations recovered from rounds 6, 7, and 8 ofthe selection were sequenced to identify individual functional clones.The sequences were grouped in families based on sequence similarity.From an analysis of Rounds 6, 7 and 8, 7 individual clones were selectedfor testing. Individual bacterial colonies corresponding to these cloneswere picked and plasmid isolated using QIAGEN Mini Prep Kit. Thesequences for each clone were PCR amplified using the F and R oligo ofthe library. Each full length clone was transcribed from the PCR productusing the protocol described before. The clones were gel purified andused for further analysis.

A summary of the clones tested is shown in Table 6. For simplicity, theconstant regions have been omitted from sequences C1 though C3.

D. Assaying Individual Clones for Binding

Individual clones were assayed by flow cytometry in a manner similar tothat described above for individual rounds of selection. In the case ofclones C1 through C3, fluorescent labeling of each aptamer was achievedvia hybridization to Dy650-N30S.R.OMe as described above.

As an initial assay, the binding of each aptamer to fD was assessedusing bead-immobilized fD when incubated at 100 nM for 1 hour at 37° C.As shown in FIG. 10, clones C1-C3 displayed significant levels ofbinding to fD beads. No binding was observed when similar experimentswere performed using beads bearing no target or a non-specific target,human growth factor.

E. Measurement of Apparent K_(d) on Beads

Flow cytometry was used to measure the binding affinity of eachindividual aptamer to fD. Assays were again performed as describedbefore but using serially diluted solutions of each aptamer. Followingincubation for 1 hour at 37° C., the beads were washed and fluorescencewas measured using flow cytometry and a plot of median fluorescentintensity versus aptamer concentration (FIG. 11) was used to determinethe apparent binding constant for each clone. Apparent K_(d) values wereobtained using the equation Y=Bmax*X/(KD+X). The apparent bindingconstant for each clone is also reported in Table 7. The apparentaffinity of aptamers to fD ranged from approximately 8 to 20 nM.

F. Competition Assays with Rounds or Individual Clones

Competition binding assays were performed using a clone of an anti-fDFab with an amino acid sequence of heavy chain variable region accordingto SEQ ID NO:7 and light chain variable region according to SEQ ID NO:8(hereinafter, “AFD”) to further assess binding. For the competitionassays, beads labelled with fD were first incubated with 50 nM round orindividual aptamer, in 50 μl SB1T (with ssDNA and BSA), for 30 minutesat 37° C. The beads were then washed with SB1T to remove unboundaptamers and incubated with or without 100 nM AFD for 30 minutes at 37°C. Following incubation, the beads were washed three times with SB1T,and assayed by flow cytometry (FIG. 12). These assays revealed thatbinding of AFD reduced the aptamer signal by ˜75%-˜90%, for both theRound 7 and 8 populations as well all selected aptamers. In cases whereaptamers are sufficiently outcompeted by AFD as described above, suchaptamers were presumed to be binding to the exosite or theself-inhibitory loop of fD.

TABLE 5 Selection details Target Input library protein Binding WashingRound pmoles/conc pmoles/conc buffer buffer washes #cycles NGS 0 1000pm/40 μM  40 pm/0.4 μM  SB1T SB1T  3 × 5 min 22 yes 1 25 pm/1 μM 40pm/0.4 μM  SB1T SB1T  3 × 5 min 22 yes 2 25 pm/1 μM 40 pm/0.4 μM  SB1TSB1T  3 × 5 min 20 yes 3 25 pm/1 μM 4 pm/0.04 μM SB1T SB1T  3 × 5 min 18yes 4 25 pm/1 μM 8 pm/0.08 μM SB1T SB1T 3 × 10 min 18 yes 5 25 pm/1 μM 8pm/0.08 μM SB1T SB1T 3 × 10 min 16 yes 6 25 pm/1 μM 4 pm/0.04 μM HBSSSB1T 4 × 15 min 14 yes 7 25 pm/1 μM 4 pm/0.04 μM HBSS HBSS + SB1T 4 × 15min 14 yes 8 25 pm/1 μM 4 pm/0.04 μM SB1T SB1T 4 × 15 min 12 yes

TABLE 6 Sequences of random region-derived sequences of select IDaptamers Compound SEQ ID NO. Name Sequence (5′ to 3′) SEQ ID NO: 1 C1GGGAGUGUGUACGAGGCAUUAGGCCGCCA with CCCAAACUGCAGUCCUCGUAAGUCUGCCUGmodifications GCGGCUUUGAUACUUGAUCGCCCUAGAAGC where G is 2′F and A, C andU are 2′OMe modified RNA SEQ ID NO: 2 C2 GGGAGUGUGUACGAGGCAUUAGUCCGCCGwith AAGUCUUUUGGCUCGGUUUUUUCAAGGUC modificationsGGCGGCUUUGAUACUUGAUCGCCCUAGAAGC where G is 2′F and A, C and U are 2′OMemodified RNA SEQ ID NO: 3 C3 GGGAGUGUGUACGAGGCAUUAGGCCGCCA withCCUCGUUUGAUUGCGGUUGUUCGGCCGCG modificationsGGCGGCUUUGAUACUUGAUCGCCCUAGAAGC where G is 2′F and A, C and U are 2′OMemodified RNA

TABLE 7 Affinity constant of selected rounds and aptamers generated inselection to fD Round/Clone K_(d) (nM) Rd 6 34.4 Rd 7 45.1 Rd 8 8.8 C112.2 C2 20.6 C3 8.5

Example 2. Identification of fD Inhibitors in Hemolysis Assays

In some cases, the disclosure provides for the identification ofaptamers that inhibit a function associated with fD. In some cases, theidentification of aptamers that that inhibit a function associated withfD may involve performing an alternative complement-dependent hemolysisassay. Human serum that is rendered deficient in the classicalcomplement pathway by depleting C1q may be dependent on alternativecomplement activity to lyse rabbit red blood cells, an activity that maybe dependent on fD. (Katschke, Wu, Ganesan, et al. (2012) Inhibitingalternative pathway complement activation by targeting the Factor Dexosite. J. Biol. Chem. 287, 12886-12892).

Briefly, citrated rabbit blood was centrifuged at 500×g for 5 minutes atroom temperature. The top plasma fraction was removed and the volume wasreplaced with 1× Veronal buffer containing 0.1% gelatin (prepared from5× Veronal buffer, Lonza #12-624E and 2% gelatin solution,Sigma-Aldrich, G1393). The red blood cells were washed two more times.The washed rabbit red blood cells were diluted in 1× Veronal buffer to aconcentration of 2×10⁹ cells/mL (RBCs).

In V-bottom 96-well plates the following reagents were added to a finalvolume of 250 μL: appropriate volume of 1× Veronal buffer with 0.1%gelatin, 100 μL aptamer, 30 μL of C1q-depleted human serum and 20 μlRBCs. This mixture was incubated for 25 minutes at room temperature,then the reaction was stopped by the addition of 5 μL of 500 mM EDTA.The plate was centrifuged for 5 minutes at 500×g at room temperature,then 100 μL of supernatant was removed and the extent of RBC lysis wasdetermined by measuring absorbance at 405 nm. Controls for the assaywere provided by complete RBC lysis with water in the absence ofC1q-depleted serum, and by inhibition of lysis caused by C1q-depletedserum by 100 μM small molecule fD inhibitor 3,4-dichloroisocoumarin.

C1-C3 identified in Example 1, a non-specific control oligo (C8), andone anti-fD Fab antibody fragment as described in Example 1 (AFD) wereincubated with C1q-depleted human serum to allow binding to fD presentin the serum, then assayed for the ability to inhibit fD-dependent lysisof rabbit red blood cells (FIG. 13). The endogenous concentration of fDwas expected to be about 9.6 nM in 10% C1q-depleted human serum (Loyet,Good, Davancaze et al. (2014) Complement inhibition in cynomolgusmonkeys by anti-factor D antigen-binding fragment for the treatment ofan advanced form of dry age-related macular degeneration. J. Pharm. Exp.Ther. 351, 527-537), so compounds that bound fD with significantlybetter affinity, such as less than 1 nM, were expected to bind nearlystoichiometrically to the fD present in the assay. This appeared to bethe case for AFD (FIG. 13; Table 8), which was reported to have a low pMaffinity for fD (20 pM, Loyet et al. 2014). IC₅₀ values for C1-C3, C8and AFD are depicted in Table 8.

TABLE 8 IC₅₀ values for C1-C3, C8 and AFD inhibiting alternativecomplement in human serum Aptamer AFD C1 C2 C3 C8 IC₅₀ 3.3 5.1 3.37.0 >160 (nM)

Example 3. Factor D Esterase Activity Assay

In some cases, a fD esterase activity assay may be used to test theactivity of putative anti-fD aptamers. In some cases, inhibition ofesterase activity may suggest that the anti-fD aptamer is binding to thecatalytic cleft, the associated substrate binding specificity pockets,or sterically occluding access to the active site. In some cases, anenhancement of esterase activity may suggest that the anti-fD aptamer isbinding to the exosite in a manner which causes allosteric activation,such as observed for an anti-fD Fab having an amino acid sequence ofheavy chain variable region according to SEQ ID NO:7 and a light chainvariable region according to SEQ ID NO:8. In yet other cases, no effecton esterase activity in combination with inhibition of hemolysis maysuggest that the anti-fD aptamer is binding the exosite in manner thatdoes not cause allosteric activation, or is binding to neither theexosite or catalytic cleft. Cleavage of a modified peptide substrate offD, such as Z-lys-S-Bzl, may be monitored by measuring the amount ofreduced 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB). FD may have a lowercatalytic rate than other complement proteases when using peptidethioester substrates, and one such substrate Z-lys-SBzl was found to becleaved by fD and useful as a synthetic substrate (fD is called proteinD in Kam, McRae et al. (1987) Human complement proteins D, C2, and B. J.Biol. Chem. 262, 3444-3451).

In one aspect a molecule that binds fD could block catalytic activity bybinding in the catalytic cleft to sterically prevent access of thepeptide substrate to the catalytic residues of fD (Katschke, Wu,Ganesan, et al. (2012) Inhibiting alternative pathway complementactivation by targeting the Factor D exosite. J. Biol. Chem. 287,12886-12892). In another aspect a molecule that binds fD could blockcatalytic activity by an allosteric mechanism that induces structuralchanges in the enzyme. In a further aspect, a molecule that binds fDcould bind to the fD exosite region to sterically inhibit binding of thephysiologic substrate protein fB, but not of the synthetic modifiedpeptide substrate Z-Lys-SBzl (Katschke, Wu, Ganesan, et al. (2012)Inhibiting alternative pathway complement activation by targeting theFactor D exosite. J. Biol. Chem. 287, 12886-12892).

In a further aspect where a molecule inhibits fD binding and proteolyticcleavage of fB but not Z-Lys-SBzl, the binding could be similar to howanti-factor D FAb antibody fragment binds to the exosite and induces asubtle conformational change that increases fD cleaving Z-Lys-S-Bzl(Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathwaycomplement activation by targeting the Factor D exosite. J. Biol. Chem.287, 12886-12892).

Briefly, in flat bottom 96-well plates, the following reagents wereadded to a final volume of 200 μL: 1× Veronal buffer with 0.1% gelatinand 10 mM MgCl₂; anti-fD antibody (AFD), aptamers (C1-C3, see Example 1)or a non-specific oligo control (C8); and a final concentration of fD ator within 5% of 10 nM, 20 nM, 40 nM, 80 nM, or 160 nM. After incubatingfor 10 min. at room temperature, Z-Lys-SBzl was added at or within 5% of94 μM, 188 μM, 375 μM, or 750 μM and DTNB at or within 5% of 5 μM, 20μM, or 40 μM. In some cases, fD was added at 41.7 nM, Z-Lys-SBzl at 375μM, and DTNB at 20.0 μM. The absorbance was immediately read in a platereader at 405 nm for 1.5 hours with a read every 30 seconds and a 3second plate shaking before each read.

Results of the assay are depicted in Table 9 and FIG. 14. Briefly, C3was determined to be an active site inhibitor based on having inhibitoryactivity comparable to a known active site inhibitor of fD,dichloroisocoumarin (DIC). When run in this assay under theseconditions, fD activity in this assay was reduced to 29±15.8% (mean±SD),which established that C3 was a potent fD inhibitor, operating via thecatalytic or active site cleft. The data further established that C2bound the exosite in a manner similar to that of AFD. The data alsoestablished that C1 either worked by a different mechanism of actionthan C2 and C3, or it functioned like C2 via the exosite, but did notaffect fD in exactly the same way to cause allosteric activation of fD.

TABLE 9 Impact of C1, C2, C3, C8 and AFD on fD Esterase activity.Aptamer AFD C1 C2 C3 C8 Activity (%) 195 83 147 26 105

Example 4. Identification of fD Inhibitors in Reconstituted Enzymatic fDAssay

In some cases, the disclosure provides for the identification of fDinhibitors in a reconstituted biochemical fD activity assay which iscomposed of purified proteins fD, fB, and C3b. When fD binds to thecomplex of fB and C3b (C3bB), fB is cleaved by fD into fragments Ba andBb (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathwaycomplement activation by targeting the Factor D exosite. J. Biol. Chem.287, 12886-12892). The activity of fD can be monitored by the rate of fBcleavage and Ba fragment production using an ELISA that uses an antibodythat specifically binds Ba (Quidel, A033).

The fB convertase assay mixture is 0.1% gelatin Veronal buffer and 10 mMMgCl₂ with complement proteins fD at or within 5% of 7.5 nM, 15 nM, 30nM, 60 nM, 120 nM, 240 nM (0.125 μM), factor B (fB) at 125 nM, 250 nM,500 nM, or 1 μM and C3b at 125 nM, 250 nM, 500 nM, or 1 μM andantibodies or aptamers.

In one example, the concentrations of fB and C3b are equal so they forma 1:1 complex which can then bind fD and allow enzymatically active fDto cleave fB to fragments Ba and Bb. In another example, the fB:C3bcomplex is present in 4-fold excess of fD. For example, final reactionconcentrations of fD of 125 nM and 0.5 μM aptamer (or a concentrationrange) are mixed for 15 minutes, then 0.5 μM fB and 0.5 μM of C3b areadded to the FD/inhibitor mixture and incubated for 30 minutes at 37°C., then 10 mM EDTA in 0.1% gelatin Veronal buffer is added to stop thereaction.

Example 5. Identification of Inhibitors of fD Binding to C3bB

In some aspects, the disclosure provides for the identification ofinhibitors of fD binding to fB in complex with C3b. FD is therate-limiting enzyme in the alternative complement pathway, and convertsthe proconvertases C3bB and C3b₂B to form the active C3 convertase C3bBbor the active C5 convertase C3b₂Bb (Katschke et al 2012). For surfaceplasmon resonance (SPR) to detect fD in a stable complex with fB, insome cases, catalytically inactive fD (S195A) can be used so that itdoes not cleave the fB upon binding to the fB:C3b complex (Katschke, Wu,Ganesan, et al. (2012) Inhibiting alternative pathway complementactivation by targeting the Factor D exosite. J. Biol. Chem. 287,12886-12892). In other cases wild type fD can be catalyticallyinactivated by the covalent inhibitor 3,4-dichloroisocoumarin (DIC)(Harper, Hemmi, Powers (1985) Reaction of serine proteases withsubstituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a newgeneral mechanism based serine protease inhibitor. Biochemistry 24,1831-1841).

When C3b is amine-coupled to a CM5 chip, SPR detects binding of fB toC3b as increased mass, and binding of fD to the resultant C3b:fB complexas a further increase in mass. fB, 3,4-dichloroisocoumarin (DIC)inactivated S195A fD, and fD binding compounds in assay buffer (Veronalbuffer, 1 mM NiCl₂, and 0.05% surfactant P-20) are flowed over the SPRchip at a flow rate of 10, 20, 30, 40, 50, or 60 μL/min, 90 μL. fB isflowed over the immobilized C3b at 0.25, 0.5, 1, 2, or 4 μM, then fB andfD are co-injected at 0.25, 0.5, 1, 2, or 4 μM fB and DIC-inactivated fDat 2-fold dilutions concentration range of 7.8 nM to 8 μM. In somecases, the flow rate is 30 μL/min and the fB concentration is 1 μM, andcomplexes formed are allowed to dissociate in assay buffer for 5minutes.

In one example, fD binding compounds are co-injected with a mixture offB and fD. For example, 1 μM fB and 1 μM 3,4-dichloroisocoumarin(DIC)-inactivated fD are co-injected with aptamers at a 2-fold dilutionrange of 1 μM to 128 μM. In one aspect, the fD binding compounds areaptamers that bind fD and prevent fD binding to fB:C3b as determined bya reduced mass detected by SPR.

Example 6. Inhibition of fD in Cell-Based Model Complement Pathology inStargardt Disease

Retinal pigment epithelial (RPE) cells undergo cell death early duringthe progress of Stargardt disease, and evidence points toward theinvolvement of the alternative complement pathway (AP) in RPE cell death(Berchuck, Yang, et al (2013) All-trans-retinal (atRal) sensitizes humanRPE cells to alternative complement pathway-induced cell death. InvestOphthalmol Vis Sci 54, 2669-2677). ARPE-19 cells are a spontaneouslyarising RPE cell line derived from the normal eyes of a 19-year-oldmale. The ARPE-19 cell line, established using the cuboidal basal celllayer cultured in specific culture media, expresses the RPE-specificmarkers cellular retinaldehyde binding protein and RPE-65.

Stargardt disease is a hereditary juvenile macular degeneration thatoccurs in patients with homozygous mutations in the ABCA4 genes, whichencode a protein that is responsible for removal of bisretinoidfluorophores, which can include N-retinylidene-N-retinyethanolamine(A2E), all-trans-retinal and related photo-oxidation products of vitaminA aldehyde which together constitute lipofuscin from photoreceptor cells(Molday (2007) ATP-binding cassette transporter ABCA4: molecularproperties and role in vision and macular degeneration. J. BioenergBiomembr 39, 507-517). An ABCA4 and RDH8 mouse model of Stargardtdisease presents with retinal pathology caused by accumulated atRal, andABCA4 mutations are present in 16% of AMD patients, suggesting thatelevated atRal may contribute to Stargardt disease and AMD diseaseprogression (Berchuck et al 2013).

Mechanistically, atRal decreased expression of CD46 and CD59 on RPEcells in vitro, which increased susceptibility to cell lysis mediated byalternative complement in response to anti-RPE antibody binding to theRPE cell membranes (Berchuck et al 2013).

In some cases, the disclosure provides for the identification of fDinhibitors that inhibit alternative complement-mediated lysis of humanretinal pigmented epithelial cells. Briefly, human RPE cells (ARPE-19cells, ATCC, Manassas, Va., USA) are grown in 1:1 mixture (vol/vol) ofDulbecco's modified Eagle's and Ham's nutrient mixture F-12;(Invitrogen-Gibco, Carlsbad, Calif., USA), non-essential amino acids 10mM, 0.37% sodium bicarbonate, 0.058% L-glutamine, 10% fetal bovineserum, and antibiotics (penicillin G 100 U/mL, streptomycin sulfate 0.1mg/mL, gentamicin 10 μg/mL, amphotericin-B 2.5 μg/mL). Cells areincubated at 37° C. in 5% CO2 and 95% relative humidity.

ARPE-19 cells are plated on six-well plates for determining cellviability in an in vitro model of Stargardt disease. 5×105 cells in 2 mLof culture media per well are plated and incubated in standardconditions for 24 hours. To sensitize cells to complement mediated lysisby atRal, ARPE-19 cells are treated with atRal for 90 minutes or 24hours. To activate the fD-dependent alternative complement pathway,cells are incubated with 24% sheep anti-RPE antibody for 30 minutes andthen treated with 6% C1q-depleted human serum. After 90 minutes at 37°C., the supernatant is collected in a 96-well plate and replaced withfresh medium. LDH release is measured in the supernatant using aCytotoxicity Detection Kit. The effect of fD-neutralizing aptamers isdetermined in the AP-induced cytotoxicity assay using defined doses(control—no drug, ½×, 1×, 2× and 10×) of all drugs.

Example 7. Treatment of Geographic Atrophy with Anti-fD Aptamer

In this example, a patient is diagnosed with geographic atrophysecondary to AMD. The patient is treated with a therapeuticallyeffective dose of a PEGylated-anti-fD aptamer by intravitrealadministration. The aptamer targets the exosite of fD and preventsbinding and cleavage of the C3bB complex. The patient is treated onceevery 4 weeks or once every 8 weeks. After six months of treatment, oneyear of treatment, and every six months thereafter, the patient isassessed for stabilization of geographic atrophy. The patient showssignificantly greater stabilization when compared to an untreatedpatient and comparable or greater stabilization when compared to apatient who has been treated with an anti-fD antibody fragment therapyonce every 4 weeks.

Example 8. Isolation of Active-Site Directed Aptamer Inhibitors of fD

In order to preferentially skew enrichment for sequences that utilizedthe library's engineered stem (FIG. 7B), we continued the selectiondescribed above in Example 1, and performed the thermal equilibrationstep in the presence of a 1.5 molar excess of the reverse primer(N30S.R; SEQ ID NO:6) and a forward blocking primer, complementary tothe 5′ end of the library (N30S.F-block; AATGCCTCGTACACACTCCC; SEQ IDNO:236). Two rounds of selection using this strategy were conducted(denoted 9FR and 10FR), and conditions are indicated in Table 10.

TABLE 10 Selection details Target Input library protein Binding WashingRound pmoles/conc. pmoles/conc. buffer buffer washes #cycles NGS  9FR 25pm/1 μM 4 pm/0.04 μM SB1T SB1T 4 × 15 min 20 no 10FR 25 pm/1 μM 4pm/0.04 μM SB1T SB1T 4 × 20 min 18 yes

Analysis of the sequence data obtained from round 9FR and 10FRidentified an additional unique sequence, 10FR-14 (Table 11).Subsequently, truncates of 10FR-14 based on formation of the engineeredstem of the library as designed yielded S31, and a further truncate ofS31 termed S32 (Table 11). These aptamers were synthesized chemically onan inverted dT CPG column bearing a 5′ six-carbon disulfide containinglinker. A sequence related to S32, Aptamer 15, containing a 5′six-carbon amino containing linker was also synthesized.

TABLE 11 Sequences of aptamers obtained from selection method CompoundSEQ ID NO: Name Sequence (5′ to 3′) SEQ ID 10FR-14GGGAGUGUGUACGAGGCAUUAGGCCGCCUUG NO: 10 withCCAGUAUUGGCUUAGGCUGGAAGUUUGGCGG modificationsCUUUGAUACUUGAUCGCCCUAGAAGC; where G is 2′F and A, C and U are 2′OMemodified RNA. SEQ ID S31 C6S- NO: 267 withAGGCCGCCUUGCCAGUAUUGGCUUAGGCUGG modifications AAGUUUGGCGGCUU-idT; whereG is 2′F and A, C and U are 2′OMe modified RNA; idT represents a3′ inverted deoxythymidine residue; C6S represents a 6-carbon disulfidecontaining linker. SEQ ID S32 C6S- NO: 268 withCCGCCUUGCCAGUAUUGGCUUAGGCUGGAAG modifications UUUGGCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; idT represents a 3′ inverteddeoxythymidine residue; C6S represents a 6-carbon disulfide containinglinker. SEQ ID Aptamer 15 C6NH₂- NO: 269 withCCGCCUUGCCAGUAUUGGCUUAGGCUGGAAG modifications UUUGGCGG-idT; where G is2′F and A, C and U are 2′OMe modified RNA; idT represents a 3′ inverteddeoxythymidine residue; C6NH₂ represents a 6-carbon amino containinglinker.

Example 9. Characterization of the Mechanism of Action of Stem-LoopAptamers

The inhibition profile of S31, S32 and Aptamer 15 was characterized infD dependent assays, including the alternative complement pathwaydependent hemolysis of red blood cells, the fD esterase assay, and thefully reconstituted biochemical fD convertase assay as detailed above inExamples 2-4, respectively. As shown in FIG. 15A, FIG. 15B, and FIG.15C, these aptamers (as exemplified by S32 in these figures) are quitepotent inhibitors of fD, with IC₅₀'s in the approximate 4-15 nM range inthe hemolysis assay, and further, possess a mechanism of action thatboth directly inhibits the active site of fD and inhibits substratebinding to fD. This conclusion is based on (1) their potent inhibitionof red-blood cell hemolysis mediated by the alternative complementpathway; (2) their ability to potently inhibit cleavage of the syntheticmodified peptide substrate Z-Lys-SBzl; (3) their ability to blockcleavage of C3bB to C3bBb+Ba in the convertase assay; (4) their abilityto inhibit convertase activity even in the presence of excess substrate;and (5) their ability to inhibit substrate binding to fD in the SPRassay. For the substrate competition assay (see FIG. 15C), anapproximately IC₃₀ concentration of S32 (150 nM) was added to fD (62.5nM) and the fD substrate complex C3b:fB 1:1 (1×=250 nM C3b and 250 nMfB; 2×=500 nM C3b and 500 nM fB; 4×=1 μM C3b and 1 μM C3b). Theenzymatic reaction proceeded for 8 minutes, at which time the fBcleavage product fBa fragment was quantified by a specific ELISA.Inhibition of fD activity is expressed as the percentage difference inthe amount of fBa produced by fD and the amount of fBa produced by fD inthe presence of S32 at each concentration.

Example 10. Structure Activity Relationship of Aptamers

Visual analysis of the sequences presented in Table 11 suggested thisfamily of aptamers formed a secondary structure comprising theengineered stem incorporated into the library, an asymmetric internalloop, a second stem, and a terminal loop. To better define the secondarystructure of this class of aptamers, as well as to potentially identifyfD aptamers with increased potency, a secondary selection was performedutilizing a partially randomized library consisting of 70% S32 parentalsequence+10% of the other 3 nucleotides at each position within S32,flanked by 5′ and 3′ constant regions. Four rounds of selection againstfD were conducted using this library, after which the library possesseda greater binding activity than S32 using the flow cytometry bindingassay described in Example 1, above. Clones from rounds 3 and 4 of thesecondary selection were sequenced, and the sequences obtained weremanually incorporated into the multiple sequence alignment shown inTable 12. The alignment of these sequences provides strong covariationsupport for a secondary structure consisting of a terminal first stem(S1), a first loop forming the 5′ side of an asymmetric internal loop(L1), a second stem (S2), a terminal second loop (L2), and a third loopforming the 3′ side of an asymmetric internal loop (L3), which joins thestructure to the terminal first stem (S1).

rd3-24 CCG--CAUUG CCAGU AUUGGCUUCG  GCUGG AAGUUUGC-CGG SEQ ID NO: 27rd3-01 CCGCA  UUG CCAGU AUAGGCUUAU  GCUGG AAGUUU  UGCGG SEQ ID NO: 28rd4-07 CCGCG  UUG CCAGU AUAGGCUUAG  GCUGG AAGUUU  CGCGG SEQ ID NO: 29rd3-47 -CGCA  UUG CUAGU AUUGGCUUAG  GCUAG AAGUGU  UGCG- SEQ ID NO: 30rd3-31 CUGC- CUUG CCAGU AUUGGCUUAG  GCUGG AAGUUUU -GCGG SEQ ID NO: 31rd4-18 CCGGG  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  CGCGG SEQ ID NO: 32rd4-03 CCGAC  UUG CCAGU AUUGGCGUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 33rd3-23 CCGAC  UUG CCAGU AUUGGCGUAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 34rd4-24 CCGAC  UUG CCAGU AUUGGCCUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 35rd4-32 UCGAC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 36rd3-39 -CGAC  UUG CCAG-UAUUGGCUUAGU -CUGG AAGUUU  GUCG- SEQ ID NO: 37rd4-20 CCGAC  UUG CCAGU GUUGGCUUUG  GCUGG AAGUUU  GUCGG SEQ ID NO: 38rd4-38 CCGAC  UUG CCAGU AUUGGCUUUG  GCUGG AAGUUU  GCCGG SEQ ID NO: 39rd4-13 CCGAC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 40rd4-37 CGGCC  UUG CCAGU AUUGGCUUUG  GCUGG AAGUUU  GGCCG SEQ ID NO: 41rd3-03 CGGCC  UUG CAUGU AUUGGCUCAG  GCAUG AAGUUU  GUCCG SEQ ID NO: 42rd4-25 CGUGC  UUG UCAGU AUUGGCGUUG  GCUGA AAGUUU  GCACG SEQ ID NO: 43rd3-27 CGUAC  UUG CCAGU AUUGGCGUAG  GCUGG AAGUUU  GUGCG SEQ ID NO: 44rd4-47 CGGGC  UUG CCAGU AUUAGGGUAG  GCUGG AAGUUU  GGCCG SEQ ID NO: 45rd4-12 CGGGC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCCG SEQ ID NO: 46rd3-33 GAGAC  UCG CCAGU AUAGGCUAAG  GCUGG AAGUAU  GUCUG SEQ ID NO: 47rd4-39 CUGAC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 48rd4-10 CUGAC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCAG SEQ ID NO: 49rd3-25 CUGAC  UUG CCAGU AUUAGCUUAG  GCUGG AAGUUU  GGCGG SEQ ID NO: 50rd4-16 CCGGC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 51rd4-21 CCGCC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 52rd3-20 CCGGC  UUG CCAGU AUUGGCUCAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 53rd3-41 CCGGC  UUG CCAGU AUAGGCUCAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 54rd3-40 UCGGC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 55rd3-02 CCGGC  UUG CCAGU AUUGGCUUUG  GCUGG AAGUUU  GCCGG SEQ ID NO: 56rd4-43 CCGGC  UUG CCAGU AUUGGCGUAG  GCUGG AAGUUU  GCCGG SEQ ID NO: 57rd4-34 CCGAC  UUG CCAGU AUUCGCUUAG  GCGGG AAGUUU  GCCGG SEQ ID NO: 58rd4-04 CCGAC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 59rd3-36 -CGAC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCG- SEQ ID NO: 60rd4-06 CCGAC  UUG CCAGU AAUGGCUUAG  GCUGG AAGUUU  GUCGG SEQ ID NO: 61rd3-13 CCGAC  UUG CCAGU AUUGGCGAUG  GCUGG AAGUUU  GUCGG SEQ ID NO: 62rd4-15 CCGAC  UUG CCAGU AUUGGCUUUG  GCUGG AAGUUU  GUCGG SEQ ID NO: 63rd3-16 CCGCC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUAU  GGCGG SEQ ID NO: 64rd4-01 CCACC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUGU  GGUGG SEQ ID NO: 65rd3-26 ACACC  UUG CGAGU AUUGGCUUAG  GCUCG AAGUUU  GGCGU SEQ ID NO: 66rd3-07 ACGC -UUUG CCAGU AUUGGCUUAG  GCUGG AAGUUUC -GCGU SEQ ID NO: 67rd4-23 CCGCG  UUG CCAGU AUUGGCUUUG  GCUGG AAGUUU  CGCGG SEQ ID NO: 68rd4-26 CCGC -CUUG CCAGU AUUGGCUUAG  GCUGG AAGUUUU -GCGG SEQ ID NO: 69rd4-30 CCGCC  UUG CCAGU AUUGGCUCAG  GCUGG AAGUUU  GGCGG SEQ ID NO: 70rd4-41 CCGCC  UUG CCAGU AUUAGCGUAG  GCUGG AAGUUU  GGCGG SEQ ID NO: 71rd4-11 CCGCC  UUG CAAGU AUUGGCGUUG  GCUUG AAGUUU  GGCGG SEQ ID NO: 72rd3-43 UCGCC  UUG CAUGU AUUGGGUUUG  GCAUG AAGUUU  GGCGG SEQ ID NO: 73rd3-28 CCGCC  UUGC-AAGU AUAGGCUUAG  GCUU- AAGUUU  GGGGG SEQ ID NO: 74rd3-34 CAGCC  AUG CAAGU AUUGGCUUAG  GCUUG AAGUUC  GGCUG SEQ ID NO: 75rd4-19 CAGCC  GUG CUAGU AUUGGCAUUG  GCUAG AAGUUA  GGCUG SEQ ID NO: 76rd3-44 CGGGC  UUG CCAGU AUUGGCGUUG  GCUGG AAGUUU  GCCCG SEQ ID NO: 77rd3-14 UGGGC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GCCCG SEQ ID NO: 78rd3-15 -GGGC  UUG CCAGU AUUGGCUUAG  GCUGG AAGUUU  GUCC- SEQ ID NO: 79

This multiple sequence alignment may define key features of these activesite-directed inhibitors of fD. For example, the fD inhibitor mayinclude: (1) a terminal stem (S1). The terminal stem may have 3-8 basepairs. The fD inhibitor may further include: (2) an asymmetric internalloop, the 5′ side of which (L1) may range from 3-5 nucleotides. In somecases, the first loop may have a minimal consensus sequence of 5′DUG 3′,where D is A, G, or U. The fD inhibitor may further include: (3) asecond stem (S2). The second stem may have 4-5 base pairs. In somecases, the second stem is not highly conserved in sequence but may havea terminal U-G base pair adjacent to L2. In other cases, the second stemmay have a terminal C-G base pair adjacent to L2. In yet other cases,the base pair at the terminus of the second stem adjacent to L2 may beany base pair. The fD inhibitor may further include: (4) a terminal loop(L2). In some cases, L2 may be 10 to 11 nucleotides in length. In somecases, L2 may have a minimal consensus sequence of 5′ DWWVGCBHWG 3′ (SEQID NO:319), where D is A, G, or U; W is A or U, V is A, C, or G, B is C,G or U and H is A, C or U. In some examples, L2 may have a U atnucleotide position 2 of L2, a U at nucleotide position 3 of L2, orboth. The fD inhibitor may further include: (5) the 3′ side of theasymmetric internal loop (L3). In some cases, L3 has 6-8 nucleotides. Insome cases, L3 has a consensus sequence of 5′ AAGUKN 3′, where K is G orU and N is any nucleotide. The consensus secondary structure of thisfamily of active-site directed inhibitors of fD is presented in FIG. 1.

The structure activity relationship for this family of active-sitedirected fD inhibitors was further probed by selective substitution of3-carbon spacers (C3) for each nucleotide in Aptamer 15, beginning withthe U residue at the first position of loop 1 (position 6 excluding thehexylamino linker) and proceeding to the U residue at the 3′ end of loop3 (position 34 excluding the hexylamino linker). These single C3 linkersubstituted molecules were assayed in the hemolysis assay to interrogatethe importance of the sugar and base identity at each substitutedposition. As can be seen in FIG. 16, substitution at U6 and U7 with C3led to a modest to moderate reduction in activity, whereas substitutionof G8 with C3 resulted in large loss of fD inhibitory activity. Thisdata is consistent with the sequence alignment presented above, in whichthe identity of the sequence within loop 1 is an important feature ofthe aptamer structure. Substitution of nucleotides within stem 2 with C3linkers, which are not capable of forming base-pairing interactions, ledto a complete to near complete loss of activity for each such aptamer,confirming the formation of stem 2 as a base-paired region within theaptamer structure. Substitution of positions within loop 2 with C3linkers demonstrated the importance of the nucleotide residues atpositions 14-17 (first 4 nucleotides of loop 2) and 23, whereasnucleotides at position 18-22 could be replaced with a C3 linker withoutloss of activity, indicating the presence of nucleotides at thesepositions is non-essential for fD inhibition. Substitution ofnucleotides at 29-32 with C3 linkers led to a complete loss of activity,demonstrating the importance of nucleotides at these residues, whereasthe C3 substitution at positions 33 and 34 was well tolerated.

Alignment of the sequences obtained from the secondary selection,combined with the results of the C3 scanning study, provide very strongevidence for the formation of the proposed secondary structure as wellas key specific sequence elements required for fD inhibition. To furtherrefine our understanding of the sequence activity relationship of thisaptamer class for fD inhibition, a series of terminal sequencetruncations, internal deletions and linker substitutions as shown inTable 13 were designed and tested for fD inhibitory activity in thealternative complement pathway dependent hemolysis assay.

TABLE 13 fD Aptamer Sequences Aptamer SEQ ID NO: Number Sequence (5′ to3′) SEQ ID NO: 270 with Aptamer 16 C6NH₂- modificationsCGCCUUGCCAGUAUUGGCUUAGGCU GGAAGUUUGGCG-idT SEQ ID NO: 271 with Aptamer17 C6NH₂- modifications GCCUUGCCAGUAUUGGCUUAGGCUG GAAGUUUGGC-idT SEQ IDNO: 272 with Aptamer 18 C6NH₂- modifications CCGCCUUGCAGUAUUGGCUUAGGCUGAAGUUUGGCGG-idT SEQ ID NO: 273 with Aptamer 19 C6NH₂- modificationsCCGCCUUGCCAGAUUGGCUUAGCUG GAAGUUUGGCGG-idT SEQ ID NO: 274 with Aptamer20 C6NH₂- modifications CCGCCUUGAAGUAUUGGCUUAGGCU UAAGUUUGGCGG-idT SEQID NO: 275 with Aptamer 21 C6NH₂- modificationsCCGCCUUGCCAGUAUUGGGCUGGAA GUUUGGCGG-idT SEQ ID NO: 276 with Aptamer 22C6NH₂- modifications CCGCCUUGCCAGUAUUGGCGGCUGG AAGUUUGGCGG-idT SEQ IDNOs: 154 and Aptamer 23 C6NH₂-CCGCCUUGCCAGUAUUG[I- 318 withmodifications 18]GGCUGGAAGUUUGGCGG-idT SEQ ID NOs: 155 and Aptamer 24C6NH₂-CCGCCUUGCCAGUAUUG[I- 318 with modifications9]GGCUGGAAGUUUGGCGG-idT SEQ ID NOs: 156 and Aptamer 25C6NH₂-CCGCCUUGCCAGUAUUGGC[I- 318 with modifications9]GGCUGGAAGUUUGGCGG-idT SEQ ID NO: 277 with Aptamer 26 C6NH₂-modifications CCGCCUUGCCAGUAUUGGCUUAGGC UGGAAGUGGCGG-idT SEQ ID NO: 278with Aptamer 27 C6NH₂- modifications CCGCCUUGCCAGUAUUGGCUUAGGCUGGAAGUUGGCGG-idT SEQ ID NO: 159 with Aptamer 28 C6NH₂- modificationsCCGCCUUGCCAGUAUUGGCUUAGGC UGGAAGU[I-6]GGCGG-idT where G is 2′F and A, Cand U are 2′OMe modified RNA; idT represents a 3′ inverteddeoxythymidine residue; [I-18] represents a hexaethylene glycol spacer;[I-9] represents a 9-carbon spacer; and [I-6] represents a 6-carbonspacer.

Aptamers 16-28 were evaluated for fD inhibitory activity at 100 and 10nM using the alternative complement dependent hemolysis assay. The datapresented in FIG. 17 demonstrate that stem 1 tolerates reduction in sizeto a 3 or 4 base pair stem. Further, loop 2 tolerates reduction in sizeto 5 or 7 nucleotides when non-nucleotidyl spacers are used tosubstitute for deleted residues, but does not tolerate a reduction insize to 5 or 7 nucleotides in the absence of additional spacersubstitutions. Reduction of stem 2 from 5 to 4 base pairs does notappear to be tolerated, although the sequencing results from thesecondary selection indicate a stem 2 of 4 base pairs is expected to befunctional in certain sequence contexts. While loop 3 toleratessubstitution of C3 linkers for U33 or U34, reduction of this loop to 4or 5 nucleotides is not tolerated, nor is substitution of both U33 andU34 with a 6-carbon spacer.

In parallel to designing aptamers based on the C3 linker substitutionand multiple sequence alignment data, variant sequences isolated in thesecondary selection were chosen to probe the importance of primary andsecondary structural features for inhibition of fD activity (Table 14).

TABLE 14 fD Aptamer Sequences Aptamer SEQ ID NO. Number Sequence (5′ to3′) SEQ ID NO: 279 with Aptamer 33 C6NH₂- modificationsCAGCCAUGCAAGUAUUGGC UUAGGCUUGAAGUUCGGCU G-idT SEQ ID NO: 280 withAptamer 34 C6NH₂- modifications CAGCCGUGCUAGUAUUGGC AUUGGCUAGAAGUUAGGCUG-idT SEQ ID NO: 281 with Aptamer 35 C6NH₂- modificationsCCGCCUUGCGAGUAAUGGC GUAGGCUCGAAGUUGGGCG G-idT SEQ ID NO: 282 withAptamer 36 C6NH₂- modifications CCGACUUGCCAGUGUUGGC UUUGGCUGGAAGUUUGUCGG-idT SEQ ID NO: 283 with Aptamer 37 C6NH₂- modificationsCAGUCUUGCGAGUUUGGCU AAGCUUCGAAGUUUGGCU G-idT SEQ ID NO: 284 with Aptamer38 C6NH₂- modifications CUGCCUUGCGGGUAUUGGC GUUGGCCCGAAGUUUGGCU G-idTSEQ ID NO: 285 with Aptamer 39 C6NH₂- modifications CCGACUUGCCAGUAUUGGCGAUGGCUGGAAGUUUGUCG G-idT SEQ ID NO: 286 with Aptamer 40 C6NH₂-modifications CGCAUUGCUAGUAUUGGCU UAGGCUAGAAGUGUUGCG- idT where G is 2′Fand A, C and U are 2′OMe modified RNA, and idT represents a 3′ inverteddeoxythymidine residue.

Aptamers 33-40 were evaluated for affinity to fD using the flowcytometric bead-immobilized fD binding assay (see Example 1) incompetition format using S32 as the fluorescently labeled aptamer.Briefly, fluorescently labeled (Dylight® 650) S32 at 200 nM was combinedwith 0, 100, 200 or 400 nM unlabeled aptamer competitor and subsequentlyincubated with bead immobilized fD. Aptamers 33-40 were also evaluatedfor fD inhibitory activity at 100 nM and 10 nM using the alternativecomplement dependent hemolysis assay. The data in FIG. 18, FIG. 19A, andFIG. 19B demonstrate that the identity of the base pairs within stem 1is not critical to fD inhibitory activity, as Aptamers 33, 34, 36, 38and 40, each of which contains base pair substitutions within stem 1,retain fD inhibitory activity. Further, the data reinforces that thelength of stem 1 can vary, as aptamers such as Aptamer 38, which has a 3base pair stem 1 (note the U-U mispair within this stem yields a 3 basepair stem) retains potent fD inhibitory activity, as does Aptamer 40,which has a 4 base pair stem 1. The data in FIG. 18, FIG. 19A, and FIG.19B further demonstrate that the identity of the first nucleotide ofloop 1 is not critical to fD inhibitor activity, as Aptamer 33(5′-AUG-3′) and Aptamer 34 (5′-GUG-3′) both retain fD inhibitoryactivity, consistent with the proposed consensus sequence for this loop.The data in FIG. 18, FIG. 19A, and FIG. 19B also demonstrate that theidentity of base pairs within stem 2 is not critical to fD inhibitoryactivity. Aptamers 33, 34, 36, 38, and 40, each of which contains basepair substitutions within stem 2, retain fD inhibitory activity. Thedata in FIG. 18, FIG. 19A and FIG. 19B further confirm the tolerance forvariability in the sequence of loop 2, consistent with the proposedconsensus structure. Of the sequences tested, only Aptamer 37 appearedto be fully inactive, which could be due to the number of changespresent in this molecule, including the length of stem 2, the sequenceof stem 1, and the length and identity of residues in loop 2. The datain FIG. 18, FIG. 19A, and FIG. 19B also demonstrate that loop 3,particularly at the 5^(th) and 6^(th) position of this loop, can bevariable, as Aptamer 33 (5′-AAGUUC-3′), Aptamer 34 (5′-AAGUUA-3′) andAptamer 35 (5′-AAGUUG-3′) all retain fD inhibitory activity, as doesAptamer 40 (5.′-AAGUGU-3′), consistent with the proposed secondarystructure. The IC₅₀ of select aptamers was determined in the alternativecomplement dependent hemolysis assay, and is shown in Table 15. Severalof these aptamers, in particular Aptamers 35, 38, 39, and 40 (see FIG.20A, FIG. 20B, FIG. 20C, and FIG. 20D), appear to be more potentinhibitors of fD than Aptamer 15, indicating the set of nucleotidesubstitutions within these aptamers may improve their function. Aptamer38 has about 80% sequence identity to Aptamer 15, and Aptamer 39 hasabout 77% sequence identity to Aptamer 15. To better assess this, a morethorough competition binding analysis was performed on two of theseclones. For these assays, fluorescently labeled (Alexa Fluor® 647)Aptamer 15 at 200 nM was incubated with bead-immobilized fD in thepresence of unlabeled Aptamers 15, 38 and 39. The data in FIG. 21demonstrate that Aptamer 38 and Aptamer 39 possess between a 2.5 and 7.7fold improvement in binding affinity when compared to Aptamer 15 in acompetition binding assay. Together these data further support theassertion that the set of nucleotide substitutions within Aptamer 38 andAptamer 39 may improve their function.

TABLE 15 IC₅₀ values for various fD aptamers Aptamer Number IC₅₀ (nM)Aptamer 33 11 Aptamer 34 6.9 Aptamer 35 5.4 Aptamer 36 8.2 Aptamer 385.5 Aptamer 39 3.1 Aptamer 40 5.8

Example 11. Refinement of Structure Activity Relationship of Aptamers

To further refine and interrogate the secondary structure determined inExample 10, two initial approaches were taken. First, structural motifswaps amongst the sequences identified in the secondary selection weredesigned to assess the robustness of the secondary structure. Second,libraries obtained in the secondary selection were subjected to deepsequencing to obtain a greater number of sequences to test andinterrogate the robustness of the consensus sequences described inExample 10.

If the stem-loop secondary structure proposed for the active-sitedirected fD aptamers is accurate, aptamers composed of secondarystructure elements such as stems or loops from one aptamer shouldgenerally be able to be substituted with equivalent sequences comprisingthe secondary structure elements from other aptamers of this family.Further, secondary structure elements of a given aptamer sequence shouldbe able to be modified per the consensus and retain fD inhibitoryactivity. Therefore, new aptamers composed of secondary structureelements from various aptamers described in this application weredesigned and their activity assessed in the alternative complementdependent hemolysis assay described in Example 2. The following stem andloop swaps were constructed and tested, with sequences and activity dataprovided in Table 16: Aptamer 53 composed of Aptamer 39 with a 4 basepair Stem 1 (S1); Aptamer 54 composed of Aptamer 39 with Stem 2 (S2) ofAptamer 38; Aptamer 55 composed of Aptamer 39 with a 4 base pair Stem 1(S1), and Stem 2 (S2) of Aptamer 38; Aptamer 56 composed of Aptamer 39with Stem 1 (S1) of Aptamer 38; Aptamer 57 composed of Aptamer 38 withLoop 2 (L2) of Aptamer 39; Aptamer 58 composed of Aptamer 39 with Stem 1(S1) from Aptamer 17; Aptamer 59 composed of Aptamer 38 with Stem 1 (S1)from Aptamer 39; and Aptamer 60 composed of Aptamer 39 with Stem 1 (S1)from Aptamer 16. As provided in Table 16, each of these aptamers, withthe exception of Aptamer 58, possessed fD inhibitory activity, providingstrong support for the secondary structure of this class of aptamers. Asdescribed in Example 12, for Aptamer 58, it is likely that the sequenceof this 3 base pair stem is sub-optimal in the context of the Aptamer 39Loop 1 (L1) sequence. Nonetheless, the inhibitory activity of Aptamers53, 55, 56, and 60 support that Stem 1 (S1) can range from 3 to 5 basepairs.

The highest frequency sequence identified from deep sequencing of thesecondary selection is Aptamer 88. As provided in Table 16, Aptamer 88provides potent fD inhibitory activity supporting sequence variationwithin Stem 1 (S1).

Sequences obtained from the deep sequencing analysis of the secondaryselection provided a number of variants to interrogate the Loop 2 (L2)consensus of 5′ DWWVGCBHWG 3′ (SEQ ID NO:319), where D is A, G, or U; Wis A or U, V is A, C, or G, B is C, G or U and H is A, C or U. Thefollowing Loop 2 (L2) sequence variants identified via deep sequencingwere constructed, and their activity was assessed in the alternativecomplement dependent hemolysis assay described in Example 2 (Nucleotidenumbering refers to the nucleotide position within Loop 2 (L2) of theconsensus secondary structure): Aptamer 64 where V at position 4 ispresent as A; Aptamer 66 where B at position 7 is present as C; Aptamer67 where W at position 3 is present as A and position 10 is U and notpresent as G as in the consensus; Aptamer 68 where B at position 7 ispresent as G, H at position 8 is present as C, and W at position 9 ispresent as U; Aptamer 69 where B at position 7 is present as G, and H atposition 8 is present as C; Aptamer 71 where B at position 7 is presentas G, and H at position 8 is present as A; Aptamer 72 where position Hat position 8 is present as C, and W at position 9 is present as U. Asprovided in Table 16, substitutions at Loop 2 (L2) positions 3, 4, 7, 8,9 which fall within the consensus 5′ DWWVGCBHWG 3′ (SEQ ID NO:319)possess anti-fD activity, confirming the consensus primary sequenceproposed for loop 2 (L2). Further, the lack of anti-fD inhibitoryactivity for Aptamer 67 provides additional support that position 10does not tolerate nucleotides other than G.

TABLE 16 fD Aptamer Sequences Aptamer SEQ ID NO: Number Sequence (5′ to3′) Activity SEQ ID Aptamer 53 C6NH₂- ++++ NO: 287 withCGACUUGCCAGUAUUGGCGAUGGCUG modifications GAAGUUUGUCG-idT SEQ ID Aptamer54 C6NH₂- +++ NO: 288 with CCGACUUGCGGGUAUUGGCGAUGGCC modificationsCGAAGUUUGUCGG-idT SEQ ID Aptamer 55 C6NH₂- +++ NO: 289 withCGACUUGCGGGUAUUGGCGAUGGCCC modifications GAAGUUUGUCG-idT SEQ ID Aptamer56 C6NH₂- ++ NO: 290 with CUGCCUUGCCAGUAUUGGCGAUGGCU modificationsGGAAGUUUGGCUG-idT SEQ ID Aptamer 57 C6NH₂- +++ NO: 291 withCUGCCUUGCGGGUAUUGGCGAUGGCC modifications CGAAGUUUGGCUG-idT SEQ IDAptamer 58 C6NH₂- − NO: 292 with GCCUUGCCAGUAUUGGCGAUGGCUGGmodifications AAGUUUGGC-idT SEQ ID Aptamer 59 C6NH₂- ++++ NO: 293 withCCGACUUGCGGGUAUUGGCGUUGGCC modifications CGAAGUUUGUCGG-idT SEQ IDAptamer 60 C6NH₂- ++++ NO: 294 with CGCCUUGCCAGUAUUGGCGAUGGCUGmodifications GAAGUUUGGCG-idT SEQ ID Aptamer 62 C6NH₂- +++ NO: 295 withCCGACUUGCCAGUAUAGGCUCAGGCU modifications GGAAGUUUGUCGG-idT SEQ IDAptamer 64 C6NH₂- + NO: 296 with CCGACUUGCCAGUAUUAGCUUAGGCUmodifications GGAAGUUUGUCGG-idT SEQ ID Aptamer 66 C6NH₂- + NO: 297 withCCGACUUGCCAGUAUUGGCCUAGGCU modifications GGAAGUUUGUCGG-idT SEQ IDAptamer 67 C6NH₂- − NO: 298 with CCGACUUGCCAGUAUAGGCUUAUGCUmodifications GGAAGUUUGUCGG-idT SEQ ID Aptamer 68 C6NH₂- +++ NO: 299with CCGACUUGCCAGUAUUGGCGCUGGCU modifications GGAAGUUUGUCGG-idT SEQ IDAptamer 69 C6NH₂- ++++ NO: 300 with CCGACUUGCCAGUAUUGGCGCAGGCUmodifications GGAAGUUUGUCGG-idT SEQ ID Aptamer 71 C6NH₂- ++++ NO: 301with CCGACUUGCCAGUAUUGGCGAAGGCU modifications GGAAGUUUGUCGG-idT SEQ IDAptamer 72 C6NH₂- + NO: 302 with CCGACUUGCCAGUAUUGGCUCUGGCUmodifications GGAAGUUUGUCGG-idT SEQ ID Aptamer 88 C6NH₂- ++++ NO: 303with CCGACUUGCCAGUAUUGGCUUAGGCU modifications GGAAGUUUGUCGG-idT where Gis 2′F and A, C and U are 2′OMe modified RNA; C6NH₂ represents ahexylamine linker; and idT represents a 3′ inverted deoxythymidineresidue. where ++++ = IC₅₀ <10 nM; +++ = IC₅₀ ~10 nM; ++ = IC₅₀ between10 and 100 nM; + = IC₅₀ of ~100 nM; − = IC₅₀ >100 nM

Example 12. Refinement of Structure Activity Relationship ofAptamers—Rational Stem Design

If the stem loop secondary structure proposed for the active-sitedirected fD aptamers is accurate, it is possible that the identity ofthe nucleotide in both stem S1 and stem S2 of the aptamer could bechanged without significant loss of aptamer function.

To this end, series of aptamer variants were designed based on the loopL2 sequences for Aptamers 15, 39, and 38 in which the sequence of S1 andS2 were altered while maintaining pairing (Table 17). These includedAptamers 90, and 94-98. Additionally, a truncate of Aptamer 38 (Aptamer91) was designed with 3 base pairs in stem S1. The stem elements (S1 andS2) from this molecule were also used to generate Aptamer 92, whichcontained L2 from Aptamer 39. As shown in Table 17, even though thesequence identity of stem S1 and S2 were significantly altered from thatof the parental sequence, anti-fD activity was maintained. The abilityto design new active sequences based upon the consensus secondarystructure by altering sequence while maintaining pairing within stems S1and S2 further supports the accuracy of the model for this genus ofaptamer.

TABLE 17 fD Aptamer Sequences SEQ ID Aptamer Design based NO: Number onSequence (5′ to 3′) Activity SEQ ID Aptamer C6NH₂- ++++ NO: 269 with 15CCGCCUUGCCAGUAU modifications UGGC

GGCUGGAA GUUUGGCGG-idT SEQ ID Aptamer Aptamer 15 C6NH₂- ++++ NO: 270with 16 CGCCUUGCCAGUAUU modifications GGC

GGCUGGAAG UUUGGCG-idT SEQ ID Aptamer Aptamer 15 C6NH₂- ++ NO: 271 with17 GCCUUGCCAGUAUUG modifications GC

GGCUGGAAGU UUGGC-idT SEQ ID Aptamer C6NH₂- ++++ NO: 285 with 39CCGACUUGCCAGUAU ++++ modifications UGGC

GGCUGGAA GUUUGUCGG-idT SEQ ID Aptamer Aptamer 39 C6NH₂- +++ NO: 287 with53 CGACUUGCCAGUAUU modifications GGC

GGCUGGAAG UUUGUCG-idT SEQ ID Aptamer Aptamer 15 C6NH₂- ++ NO: 308 with94 GC CCUUG UCC GUAUU modifications GGC

GGC G GAAAG UUUGG GC -idT SEQ ID Aptamer Aptamer 15 C6NH₂- ++ NO: 311with 97 G GCCUUGCC C GUAUU modifications GGC

GGC G GGAAG UUUGGC C -idT SEQ ID Aptamer Aptamer 39 C6NH₂- ++ NO: 309with 95 GCGG UUGC GGG UAU modifications UGGC

GGC CCG AA GUUU CCGC -idT SEQ ID Aptamer Aptamer 39 C6NH₂- ++++ NO: 312with 98 GGC CUUGCC C GUAUU modifications GGC

GGC G GGAAG UUUG GCC -idT SEQ ID Aptamer Aptamer 15 C6NH₂- − NO: 304with 90 C C G UUG UGG GUAUUG modifications GC

GGC CCA AAGU U UCGG -idT SEQ ID Aptamer Aptamer 15 C6NH₂- ++ NO: 310with 96 C CCUUGCC C GUAUUG modifications GC

GGC G GGAAGU UUGG G -idT SEQ ID Aptamer Aptamer 39 C6NH₂- + NO: 307 with93 GC CUUG AGG GUAUUG modifications GC

GGC CCU AAGU UU GGC -idT SEQ ID Aptamer Aptamer 39 C6NH₂- +++ NO: 306with 92 GC CUUGC GG GUAUUG modifications GC

GGC CC GAAGU UUG GC -idT SEQ ID Aptamer Aptamer 38 C6NH₂- ++ NO: 305with 91 GCCUUGCGGGUAUUG modifications GC

GGCCCGAAGU UUGGC-idT where G is 2′F and A, C and U are 2′OMe modifiedRNA; C6NH₂ represents a hexylamine linker; and idT represents a3′ inverted deoxythymidine residue. Bolded, italicized nucleotidesindicate differences in L2 between the sequences. Bolded, underlinednucleotides indicate differences between designed stems and parentalmolecule Activity Key: ++++ = IC₅₀ <10 nM; +++ = IC₅₀ ~10 nM; ++ = IC₅₀between 10 and 100 nM; + = IC₅₀ of ~100 nM; − = IC₅₀ >100 nM

Example 13. Optimization of fD Aptamers by 2′Ome Sugar Substitutions

2′OMe modifications are known to impart higher duplex stability and havegreater coupling efficiency during synthesis compared to 2′F-containingnucleotides. The use of these nucleotides also avoids the potential lossof the 2′F group which can happen during deprotection and exposure toheat. To probe the effect of 2′F-G to 2′OMe-G substitution on targetbinding, variants of Aptamer 15 were synthesized where 2′F-G at certainpositions was selectively substituted with 2′OMe-G (Table 18). 5variants of Aptamer 15 were synthesized—Aptamer 74 with 2′OMe-G atpositions 3, 39, 38, 36, and 35 in Stem 1 (S1); Aptamer 1721 with2′OMe-G at positions 3, 39, 38, 36, and 35 in Stem 1 (S1) and atpositions 12 and 27 in Stem 2 (S2); Aptamer 1722 with 2′OMe-G atpositions 3, 39, 38, 36, and 35 in Stem 1 (S1) and at positions 12, 27,and 28 in Stem 2 (S2); Aptamer 1723 with 2′OMe-G at positions 3, 39, 38,36, and 35 in Stem 1 (S1) and at positions 12, 24, 27, and 28 in Stem 2(S2); Aptamer 1724 with 2′OMe-G at positions 3, 39, 38, 36, and 35 inStem 1 (51), at positions 12, 24, 27, and 28 in Stem 2 (S2), at position8 in Loop 1 (L1), at positions 17, 18, and 23 in Loop 2 (L2) andposition 31 in Loop 3 (L3). These variants were assayed to determinetheir binding affinity to fD using the bead-based direct binding assaydescribed in Example 1. As shown in Table 18, 2′F-G to 2′OMe-Gsubstitutions were well tolerated in Stem 1 (S1) which was evident fromthe higher affinity of Aptamer 74 as compared to the parent Aptamer 15and other 2′OMe-G variants. This could be due to increased stability inthe Stem 1 (S1) architecture resulting from the inclusion of 2′OMe-Gs,which may improve the overall structural stability of the aptamer.Interestingly, 2′OMe-G substitutions in Stem 2 (S2) (Aptamers 1721 to1723) are tolerated, but do affect the affinity as determined by flowcytometry, suggesting a role for the 2′F-G residues within this stem.2′OMe-G substitutions were not tolerated in Loop 1, 2 or 3 regions whenall of the 2′F-G residues were modified (Aptamer 1724), clearlyindicating that 2′F-G residues in the loop regions are either crucial inmaintaining the loop structure required for binding fD or are makingdirect contacts with fD. Together these data clearly support that2′OMe-G substitutions in Stem 1 (S1) of Aptamer 15 enhance its affinityto fD and improve its fD inhibitory function.

TABLE 18 fD Aptamer Sequences with 2′OMe-G Substitutions Aptamer SEQ IDNO number Sequence K_(d) SEQ ID Aptamer 15 RNA C6NH₂- 7-10 nM NO: 269with CCGCCUUGCCAGUAUUGGCUU modifications AGGCUGGAAGUUUGGCGG-idT SEQ IDAptamer 74 RNA C6NH₂- 2.7 nM NO: 269 with CCGCCUUGCCAGUAUUGGCUUmodifications AGGCUGGAAGUUUGGCGG-idT SEQ ID Aptamer 1721 RNA C6SH- 4.5nM NO: 269 with CCGCCUUGCCAGUAUUGGCUU modificationsAGGCUGGAAGUUUGGCGG-idT SEQ ID Aptamer 1722 RNA C6SH- 11.0 nM NO: 269with CCGCCUUGCCAGUAUUGGCUU modifications AGGCUGGAAGUUUGGCGG-idT SEQ IDAptamer 1723 RNA C6SH- 45 nM NO: 269 with CCGCCUUGCCAGUAUUGGCUUmodifications AGGCUGGAAGUUUGGCGG-idT SEQ ID Aptamer 1724 RNA C6SH- <1000nM NO: 269 with CCGCCUUGCCAGUAUUGGCUU modificationsAGGCUGGAAGUUUGGCGG-idT where G is 2′F, and G, A, C and U are 2′OMemodified RNA, C6NH₂ represents a six-carbon amino containing linker,C6SH represents a six-carbon thiol linker, and idT represents a3′ inverted deoxythymidine residue.

Due to the synthetic and stability advantages of 2′OMe-Gs over 2′F-Gsand that 2′OMe-G substitutions in Stem 1 (51) improved affinity ofAptamer 74, similar substitutions were constructed in Stem 1 (S1) ofother potent fD aptamers and their activity was assessed in thealternative complement dependent hemolysis assay. The following nineaptamers with 2′OMe-Gs in Stem 1 (S1) were synthesized: Aptamer 76(2′OMe-Gs substituted in Stem 1 (S1) of Aptamer 39), Aptamer 116(Aptamer comprising loop 2 (L2) of Aptamer 39 and Stem 1 (51) of Aptamer15 with 2′OMe-Gs in Stem 1 (S1)), Aptamer 102 (4 base pair 2′OMe-G Stem1 (S1) variant of Aptamer 76), Aptamer 104 (4 base pair 2′OMe-G Stem 1(S1) variant of Aptamer 74), Aptamer 106 (Aptamer 88 with 2′OMe-G Stem 1(S1)), Aptamer 107 (4 base pair 2′OMe-G Stem 1 (S1) variant of Aptamer88), Aptamer 108 (3 base pair 2′OMe-G Stem 1 (S1) variant of Aptamer92), Aptamer 109 (4 base pair 2′OMe-G Stem 1 (S1) variant of Aptamer 98)and, Aptamer 99 (4 base pair 2′OMe-G Stem 1 (S1) variant of Aptamer 60).All of these aptamers demonstrated very high potency with IC₅₀<10 nM inalternative complement dependent hemolysis assay as shown in Table 19.This data demonstrates that 2′OMe-G substitutions in Stem 1 (S1) may bepreferred and improve the fD inhibitory activity and fD affinity of thestem-loop aptamers.

TABLE 19 fD Aptamer Sequences with 2′OMe-G Substitutions SEQ ID NOAptamer number Sequence Activity SEQ ID Aptamer 74 RNA C6NH₂- ++++ NO:269 with CCGCCUUGCCAGUAUUGGCU modifications UAGGCUGGAAGUUUGGCGG- idT SEQID Aptamer 76 RNA C6NH₂- ++++ NO: 285 with CCGACUUGCCAGUAUUGGCmodifications GAUGGCUGGAAGUUUGUCG G-idT SEQ ID Aptamer 116 RNA C6NH₂-++++ NO: 316 with CCGCCUUGCCAGUAUUGGCG modificationsAUGGCUGGAAGUUUGGCGG- idT SEQ ID Aptamer 102 RNA C6NH₂- ++++ NO: 313 withCGACUUGCCAGUAUUGGCG modifications AUGGCUGGAAGUUUGUCG- idT SEQ ID Aptamer104 RNA C6NH₂- ++++ NO: 314 with CGCCUUGCCAGUAUUGGCU modificationsUAGGCUGGAAGUUUGGCG- idT SEQ ID Aptamer 106 RNA C6NH₂- ++++ NO: 303 withCCGACUUGCCAGUAUUGGC modifications UUAGGCUGGAAGUUUGUCG G-idT SEQ IDAptamer 107 RNA C6NH₂- ++++ NO: 315 with CGACUUGCCAGUAUUGGCUmodifications UAGGCUGGAAGUUUGUCG- idT SEQ ID Aptamer 108 RNA C6NH₂- ++++NO: 306 with GCCUUGCGGGUAUUGGCGA modifications UGGCCCGAAGUUUGGC-idT SEQID Aptamer 109 RNA C6NH₂- ++++ NO: 312 with GGCCUUGCCCGUAUUGGCGmodifications AUGGCGGGAAGUUUGGCC- idT SEQ ID Aptamer 99 RNA C6NH₂- ++++NO: 294 with CGCCUUGCCAGUAUUGGCG modifications AUGGCUGGAAGUUUGGCG- idTwhere G is 2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂represents a six-carbon amino containing linker, C6SH represents asix-carbon thiol linker, and idT represents a 3′ inverted deoxythymidineresidue.

Example 14. Affinity Measures for fD Aptamers

The binding affinity of aptamers for fD was determined by surfaceplasmon resonance (SPR) on a Reichert4 SPR System, where either aptameror fD was immobilized as ligand on a solid surface and the interactantanalyte was flowed in solution over the immobilized ligand (Table 20).In brief, 14-30 μg/mL human fD in 10 mM sodium acetate pH 5.2 or pH 5.7was amine coupled to an EDC/NHS activated dextran chip surface, thenblocked with 1 M ethanolamine pH 8.5, resulting in 835 RIU immobilizedfD. 9 point, 2-fold aptamer dose response curves with 125 nM topconcentration were then generated, and K_(D) affinity values werecalculated as the ratio of the dissociation and association rateconstants (K_(D)=K_(d)/K_(a)) and are presented in Table 20. Affinitieswere similar sub- to low-nM, ranging from 0.6 nM to 4.2 nM (Table 20).

TABLE 20 fD Aptamer Sequences with 2′OMe-G Substitutions SPR AffinitySEQ ID NO. Aptamer number Sequence (nM) SEQ ID NO: 269 with 74 RNAC6NH₂- 3.7 modifications CCGCCUUGCCAGUAU UGGCUUAGGCUGGAA GUUUGGCGG-idTSEQ ID NO: 285 with 76 RNA C6NH₂- 1.6 modifications CCGACUUGCCAGUAUUGGCGAUGGCUGGAA GUUUGUCGG-idT SEQ ID NO: 313 with 102 RNA C6NH₂- 2.5modifications CGACUUGCCAGUAUU GGCGAUGGCUGGAAG UUUGUCG-idT SEQ ID NO: 314with 104 RNA C6NH₂- 3.9 modifications CGCCUUGCCAGUAUU GGCUUAGGCUGGAAGUUUGGCG-idT SEQ ID NO: 303 with 106 RNA C6NH₂- 1.7 modificationsCCGACUUGCCAGUAU UGGCUUAGGCUGGAA GUUUGUCGG-idT SEQ ID NO: 315 with 107RNA C6NH₂- 4.2 modifications CGACUUGCCAGUAUU GGCUUAGGCUGGAAG UUUGUCG-idTSEQ ID NO: 306 with 108 RNA C6NH₂- 2.7 modifications GCCUUGCGGGUAUUGGCGAUGGCCCGAAGU UUGGC-idT SEQ ID NO: 312 with 109 RNA C6NH₂- 0.6-1.9modifications GGCCUUGCCCGUAUU GGCGAUGGCGGGAAG UUUGGCC-idT SEQ ID NO: 294with 99 RNA C6NH₂- 3.1-3.6 modifications CGCCUUGCCAGUAUU GGCGAUGGCUGGAAGUUUGGCG-idT where G is 2′F, and G, A C and U are 2′OMe modified RNA,C6NH₂ represents a six-carbon amino containing linker, C6SH represents asix-carbon thiol linker, and idT represents a 3′ inverted deoxythymidineresidue.

The binding affinity of aptamers for fD was determined in solution usinga homogenous time-resolved fluorescence resonance energy transfer(TR-FRET) assay (Table 21). In brief, 2.5 nM recombinant human Factor Dwith a hexahistidine tag (SEQ ID NO:324) (His), 2.5 nM anti-HisEuropium, and varied concentrations of Dylight® 650-labeled aptamer wasadded to half-well black 96-well plates in TR-FRET buffer (50 mM MOPS,pH 7.4, 125 mM NaCl, 5 mM KCl, 0.1 mg/mL BSA, 50 μM CHAPS, 1 mM CaCl₂and 1 mM MgCl₂). After 1 hour incubation at 25° C., binding activity wasassessed using a BioTek® Cytation™ 5 plate reader. Affinity bindingconstants were calculated as the concentration at which 50% of aptameris bound using a single site binding model, with values ranging from0.66 nM to 2.5 nM (Table 21).

TABLE 21 fD Aptamer Sequences with 2′OMe-G Substitutions TR- FRETAptamer Affinity SEQ ID NO. number Sequence (nM) SEQ ID NO: 269 74 RNAC6NH₂- 2.5 with modifications CCGCCUUGCCAGUAUUGGCUUAGGCUGGAAGUUUGGCGG-idT SEQ ID NO: 285 76 RNA C6NH₂- 0.8 withmodifications CCGACUUGCCAGUAUUGGCGA UGGCUGGAAGUUUGUCGG-idT SEQ ID NO:313 102 RNA C6NH₂- 1.6 with modifications CGACUUGCCAGUAUUGGCGAUGGCUGGAAGUUUGUCG-idT SEQ ID NO: 303 106 RNA C6NH₂- 0.66 withmodifications CCGACUUGCCAGUAUUGGCUU AGGCUGGAAGUUUGUCGG-idT where G is2′F, and G, A, C and U are 2′OMe modified RNA, C6NH₂ represents asix-carbon amino containing linker, C6SH represents a six-carbon thiollinker, and idT represents a 3′ inverted deoxythymidine residue.

Example 15. SPR Complex Assembly Data

In some cases, surface plasmon resonance (SPR) may be used to assemblefD in complex with the natural substrate fB when fB is in complex withC3b (C3bB), where C3b is immobilized to a solid surface, incubated withfB to allow the C3bB complex to form, and then further incubated withenzymatically inactivated fD (*fD) to allow *fD to bind to the C3bBsubstrate without proteolytically cleaving fB. This assay may be used totest whether putative anti-fD aptamers compete for binding to fD withC3bB. In some cases, an anti-fD aptamer may bind and inhibit fD enzymeactivity by binding at or near the active site without interfering withC3bB substrate binding. In some cases, an anti-fD aptamer may bind fDand inhibit C3bB substrate binding. In some cases, an anti-fD aptamermay bind at or near the active site and inhibit enzyme activity, whilealso binding in such a manner as to inhibit C3bB substrate binding.

In one aspect, lack of inhibition of fD binding to C3bB may suggest thatthe anti-fD aptamer is binding to fD at the catalytic site withoutinterfering, or competing, with C3bB binding to fD. In one aspect,inhibition of fD binding to C3bB may suggest that the anti-fD aptamer isbinding to fD on a shared portion of the interface between fD and C3bBsuch that the aptamer interferes, or competes, with C3bB binding to fDin a substrate competitive mechanism. Given the relative proximity ofthe active site and the substrate binding exosite of fD, it is alsopossible that anti-fD aptamer is binding on a shared portion of theinterface between fD and C3bB such that the aptamer interferes, orcompetes, with C3bB binding to fD in a substrate competitive mechanism,and also blocks access to the active site of fD.

Briefly, human fD was pre-incubated with 50 μM of the covalent fDinhibitor, 3,4-dichloroisocoumarin (DIC) for 1 hour, which completelyinactivated fD esterase activity (referred to hereinafter as *fD) sothat it could form a complex with C3bB without enzymatically cleavingthe fB substrate. 25 μg/mL human C3b in 10 mM sodium acetate pH 4.0 wasamine coupled to an EDC/NHS activated dextran chip surface for 0.5-10minutes, then blocked with 1 M ethanolamine pH 8.5, resulting in800-2,242 RIU immobilized, then *ID was injected as a negative control.1 μM fB was injected for 3 minutes to allow the C3bB complex to form,then 1 μM fB preincubated with 1 μM*fD plus up to 2 μM aptamer wasinjected for 3 minutes to allow the C3bB:fD complex to form. Followingeach injection of fB/*fD/aptamer, the complexes were dissociated fromthe bound C3b with 2×60 seconds injections of 3M NaCl in 50 mM sodiumacetate pH 5.0.

Results of the assay are depicted in FIG. 22, which has the resultspresented as non-ligand channel subtracted from the C3b-ligand channel.fB was bound to immobilized C3b to form C3bB, which was further bound by*fD (fD (DCI)) as indicated by an increase in signal to form the complexC3bB:*fD. In contrast, when the experiment was repeated in the presenceof Aptamer 15, the immobilized C3b still bound fB to form C3bB, butaddition of *fD did not further increase the signal, indicating thatAptamer 15 inhibited *fD binding to the C3bB substrate. When fB plusAptamer 15 was added, there was also no increase in signal above fBalone, confirming Aptamer 15 specificity for *fD. Thus, under allconditions, fB bound to the immobilized C3b, which could then bind *fD,but the *fD binding to C3bB was inhibited by Aptamer 15, consistent withAptamer 15 binding *fD and inhibiting *fD binding to substrate C3bB.Similar results as for Aptamer 15 were observed for the other aptamersP01 and P04 (described in Example 16) (FIG. 22). Throughout the sampleruns, the immobilized C3b retained the ability to bind fB and *fD, asthe final sample injection of fB and *fD had fB and *fD both binding tothe immobilized C3b similarly to the beginning of the sample runs (FIG.22).

Aptamer inhibition of C3bB:*fD complex assembly was further demonstratedby titrating 2-fold Aptamer P04 (PEGylated Aptamer 74, described inExample 16) from 4 μM down to 31.3 nM (FIG. 23). A dose-dependentinhibition of C3bB:*fD complex assembly was observed throughout thedose-response curve, consistent with Aptamer P04 binding *fD andinhibiting *fD binding to substrate C3bB (FIG. 23). This data, incombination with the inhibition of fD esterase activity against smallsynthetic substrates (Example 9), demonstrate that this stem-loop classof anti-fD aptamer, represented by Aptamer 15, inhibits fD by at leasttwo mechanisms of action. First, this class of aptamer possesses anactive-site directed mechanism which blocks access to the active siteand/or directly inhibits fD catalytic activity, and second, it preventsassociation of its biologic substrate, C3bB, with fD.

Example 16. Pegylation of Aptamers

Potent fD inhibiting Aptamers 15, 74, 76, 102 and 106 were conjugated toa 40 kDa branched PEG to evaluate their tolerance for pegylation.Briefly, a concentrated feed solution consisting of aptamer in DMSO, 16to 25 mM borate and water was combined with a solution consisting ofseveral equivalents2,3-Bis(methylpolyoxyethylene-oxy)-1-{3-[(1,5-dioxo-5-succinimidyloxy,pentyl)amino]propyloxy} propane (for example SUNBRIGHT® GL2-400GS2) inacetonitrile, and incubated at approximately 35° C. for approximately 1hour with mixing to effect conjugation of the PEG to the amine moiety ofthe hexyl amine linker present on the 5′ terminus of the aptamer.Following the pegylation reaction, each PEG-aptamer was purified byanion exchange chromatography to collect the pegylated aptamer andremove unreacted PEG and unreacted aptamer. Anion exchange purifiedPEG-aptamers were desalted by ultrafiltration into water prior tofunctional characterization. The pegylated versions of aptamers 15, 74,76, 102 and 106 are termed aptamers P01, P04, P06, P07 and P08,respectively.

Example 17. In Vitro Characterization of Pegylated Aptamers

The potency of the PEG-aptamers P01, P04, P06, P07 and P08 compared totheir non-pegylated counterparts was determined in the alternativecomplement-dependent hemolysis assay as described in Example 2. As shownin Table 22, this class of aptamer tolerates pegylation well, with eachPEG-aptamer exhibiting a modest increase in potency compared to itsnon-pegylated counterpart as determined by their respective IC₅₀ valuesin this assay.

TABLE 22 IC₅₀ values for select PEG and non-PEG aptamers IC₅₀ nM Aptamer(mean ± sd)  15 9.4 ± 4.4 P01 6.1 ± 1.5  74 7.0 ± 1.4 P04 3.2 ± 0.8  764.2 ± 0.8 P06 4.1 ± 0.9 102 6.3 ± 0.1 P07 4.4 ± 0.6 106 5.2 ± 0.4 P084.1 ± 0.5

Example 18. In Vivo Characterization of PEG-Aptamer P01

Aptamer P01 was selected as a representative pegylated form of an activesite directed inhibitor of fD with a stem loop secondary structure tocharacterize the duration of action of this class of aptamer followingintravitreal administration to rabbits. Sixteen New Zealand Whiterabbits, two rabbits per timepoint, were treated with 1.5 mg/eye ofaptamer P01 administered by intravitreal injection. Vitreous and plasmasamples were taken at 1, 8, 24, 48, 96, 168, 240 and 336 hours postaptamer P01 administration with individual samples being obtained fromthe left and right eye of each animal at each timepoint. Vitreous andplasma samples were also obtained from 2 placebo treated animals toserve as controls for sample analysis. The duration of action of aptamerP01 was determined by measuring the anti-fD activity retained in thevitreous over time following administration using the alternativecomplement-dependent hemolysis assay. Additionally, the terminalconcentration of aptamer P01 was measured using a dual hybridizationELISA assay.

For ex vivo measurement of the retained anti-fD activity of aptamer P01following intravitreal administration, a small volume of vitreousobtained from each eye was added to normal human serum and tested in thealternative complement-dependent hemolysis assay as described in Example2. Absorbance readings obtained from treated samples in the hemolysisassay were normalized to those obtained when vitreous from controlanimals was tested in parallel in this assay to determine the percent offD inhibition observed. As shown in Table 23, inhibition of fD activityby aptamer P01 was consistent from 1 to 336 hours post administration,with essentially complete fD inhibition observed at each time point.Therefore, aptamer P01 maintains complete inhibition of fD activity inrabbits for at least 14 days following a single intravitrealadministration of 1.5 mg/eye.

TABLE 23 Percent inhibition of fD activity ex vivo followingintravitreal administration of aptamer P01 Timepoint (hr postadministration) 1 8 24 48 96 168 240 336 % fD Inhibition 96.4 96.4 95.996.2 96.3 96.6 96.4 96

To further characterize the duration of action of aptamer P01, theterminal concentration was determined by measuring the concentration ofP01 in the vitreous 336 hours post intravitreal administration. Theconcentration of aptamer P01 in the vitreous at the 336 hour timepointwas approximately 4,600 nM. This concentration is approximately 760-foldgreater than the IC₅₀ of aptamer P01 in the alternativecomplement-dependent hemolysis assay and 270-fold greater than theconcentration of complement fD in the human vitreous, which isapproximately 17 nM (Loyet, DeForge, Katschke Jr., et al. (2012)Activation of the alternative complement pathway in vitreous iscontrolled by genetics in age-related macular degeneration. Invest.Ophthalmol: Vis. Sci. 53:6628-6637). The pegylated aptamer Macugen®which has been well-studied following intravitreal administration inanimals and humans contains similar sugar modifications to aptamer P01and is conjugated to similar 2-arm branched 40 kDa PEG, and provides agood analog from which to extrapolate the expected performance ofaptamer P01 in humans. Assuming that aptamer P01 has a half-life inrabbits of at least 80 hours, similar to Macugen® (The EyeTech StudyGroup (2002) Preclinical and phase 1A clinical evaluation of ananti-VEGF pegylated aptamer (EYE001) for the treatment of exudativeage-related macular degeneration. Retina 22(2): 143-152), and thatcomplete inhibition of fD will be achieved when the aptamerconcentration is two-fold or greater than the vitreous fD concentration,aptamer P01 administered at 1.5 mg/eye would be anticipated to providecomplete inhibition of fD following intravitreal administration inrabbits for at least 30 days, the time at which the vitreousconcentration of aptamer P01 is extrapolated to be, under theseassumptions, approximately 40 nM. Macugen® has a half-life in humansfollowing intravitreal administration of approximately 10 days(“MACUGEN®, Drugs at FDA;https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021756s0181b1.pdf).If the in vivo performance of aptamer P01 performs like Macugen® andexhibits a similar approximately 3-fold increase in half-life in humansas compared to rabbits, one would anticipate a duration of action ofapproximately 90 days or greater for aptamer P01 following intravitrealadministration of 1.5 mg/eye. Minimally, one would anticipateintravitreal administration of 1.5 mg/eye of P01 to provide atherapeutic effect for at least 60 days, and potentially up to 120 days.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. An aptamer comprising a nucleic acid sequencethat selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein said third loop comprises 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein said first loop has fewer nucleotides than said second loop. 2.The aptamer of claim 1, wherein said third loop is connected to saidfirst base-paired stem.
 3. The aptamer of claim 1, wherein said firstloop has from 1 to 10 nucleotides, non-nucleotidyl spacers, or acombination thereof.
 4. The aptamer of claim 1, wherein said second loopcomprises at least 6 nucleotides, non-nucleotidyl spacers, or acombination thereof.
 5. The aptamer of claim 1, wherein said third loophas from 6 to 8 nucleotides, non-nucleotidyl spacers, or a combinationthereof.
 6. The aptamer of claim 1, wherein said first base-paired stemhas from 2 to 10 base pairs.
 7. The aptamer of claim 1, wherein saidsecond base-paired stem has from 2 to 10 base pairs.
 8. The aptamer ofclaim 1, wherein said third loop comprises at least 4 nucleotides and upto 2 non-nucleotidyl spacers.
 9. The aptamer of claim 1, wherein saidthird loop comprises at least 6 nucleotides.
 10. The aptamer of claim 1,wherein: a) said first base-paired stem is adjacent to said first loop;b) said second base-paired stem is adjacent to said first loop, saidsecond loop, and said third loop; or c) said first base-paired stem isadjacent to said first loop and said second base-paired stem is adjacentto said first loop, said second loop, and said third loop.
 11. Theaptamer of claim 1, wherein said aptamer is an RNA aptamer or a modifiedRNA aptamer.
 12. The aptamer of claim 1, wherein said aptamer is a DNAaptamer or a modified DNA aptamer.
 13. The aptamer of claim 1, whereinsaid aptamer comprises one or more modified nucleotides.
 14. The aptamerof claim 13, wherein said one or more modified nucleotides comprises a2′F-modified nucleotide, a 2′OMe-modified nucleotide, or a combinationthereof.
 15. The aptamer of claim 1, wherein said stem-loop structurehas exactly two base-paired stems.
 16. The aptamer of claim 1, whereinsaid aptamer is an RNA aptamer comprising nucleotides having ribose in aβ-D-ribofuranose configuration.
 17. The aptamer of claim 1, wherein saidaptamer blocks an active site of fD.
 18. The aptamer of claim 1, whereinsaid aptamer blocks a substrate-binding exosite of fD.
 19. The aptamerof claim 1, wherein said aptamer blocks both an active site and asubstrate-binding exosite of fD.
 20. The aptamer of claim 1, whereinsaid aptamer inhibits a function associated with fD.
 21. The aptamer ofclaim 1, wherein said nucleic acid sequence comprises from 30 to 90nucleotides, non-nucleotidyl spacers, or a combination thereof.
 22. Theaptamer of claim 1, wherein said aptamer selectively binds to an activesite of fD with a K_(d) of less than about 50 nM.
 23. The aptamer ofclaim 1, wherein said aptamer inhibits fD in an alternative complementdependent hemolysis assay with an IC₅₀ of less than about 50 nM.
 24. Theaptamer of claim 1, wherein said aptamer inhibits fD in a fD convertaseassay with an IC₅₀ of less than about 50 nM.
 25. The aptamer of claim 1,wherein said aptamer inhibits fD activity in an esterase activity assay.26. The aptamer of claim 1, wherein said aptamer binds to fD with aK_(d) of less than about 50 nM and inhibits fD in an alternativecomplement dependent hemolysis assay with an IC₅₀ of less than about 100nM.
 27. The aptamer of claim 1, wherein: (i) said aptamer is conjugatedto a polyethylene glycol (PEG) molecule; (ii) said aptamer comprisesmodified RNA; and (iii) said aptamer selectively binds to an active siteof fD with a K_(d) of less than about 50 nM.
 28. An aptamer comprising anucleic acid sequence that selectively binds to complement factor D (fD)and having a stem-loop secondary structure comprising, in a 5′ to 3′direction, a first base-paired stem, a first loop, a second base-pairedstem, a second loop, and a third loop, wherein said third loop comprises6 or more nucleotides, non-nucleotidyl spacers, or a combinationthereof, and wherein said second loop comprises more than 5 nucleotides,non-nucleotidyl spacers, or a combination thereof.
 29. An aptamercomprising a nucleic acid sequence that selectively binds to complementfactor D (fD) and having a stem-loop secondary structure comprising, ina 5′ to 3′ direction, a first base-paired stem, a first loop, a secondbase-paired stem, a second loop, and a third loop, wherein said thirdloop comprises 6 or more nucleotides, non-nucleotidyl spacers, or acombination thereof, and wherein said third loop is adjacent to saidfirst base-paired stem.
 30. An aptamer comprising a nucleic acidsequence that selectively binds to complement factor D (fD) and having astem-loop secondary structure comprising, in a 5′ to 3′ direction, afirst base-paired stem, a first loop, a second base-paired stem, asecond loop, and a third loop, wherein said third loop comprises 6 ormore nucleotides, non-nucleotidyl spacers, or a combination thereof, andwherein said first base-paired stem has no more than 5 base pairs.